Cell cloning Cloning unicellular organisms

Cloning cell-line colonies using cloning rings Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media. A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth. Cloning stem cells Somatic-cell nuclear transfer, known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning." The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.[6]

Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimers. The process begins by taking out the nucleus (containing the DNA) from an egg cell and putting in it a nucleus from the adult cell to be cloned. In the case of someone with Alzheimers disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient. The embryo will then form a blastocyst which has the potential to form/become any cell in the body. The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the oocyte is inserted into the donors somatic cell nucleus. The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells. The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use. The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm. This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current. This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals. SCNT is seen as a good method for producing agriculture animals for food consumption. It successfully cloned sheep, cattle, goats, and pigs. Another benefit is SCNT is seen as a solution to clone endangered species that are on the verge of going extinct. However, stresses placed on both the egg cell and the introduced nucleus are enormous, leading to a high loss in resulting cells. For example, the cloned sheep Dolly was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood. As the procedure currently cannot be automated, and has to be performed manually under a microscope, SCNT is very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg is also far from being well-understood.

In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus. Organism cloning Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants and some insects. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation, has been common practice in the horticultural world for hundreds of years. Horticultural The term clone is used in horticulture to refer to descendants of a single plant which were produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation. Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies naturally. Parts of an individual plant may become detached by fragmentation and grow on to become separate clonal individuals. A common example is in the vegetative reproduction of moss and liverwort gametophyte clones by means of gemmae. Some vascular plants e.g. dandelion and certain viviparous grasses also form seeds asexually, termed apomixis, resulting in clonal populations of genetically identical individuals. Parthenogenesis Clonal derivation exists in nature in some animal species and is referred to

as parthenogenesis (reproduction of an organism by itself without a mate). This is an asexual form of reproduction that is only found in females of some insects, crustaceans and lizards. The 3

growth and development occurs without fertilization by a male. In plants, parthenogenesis means the development of an embryo from an unfertilized egg cell, and is a component process of apomixis. In species that use the XY sex-determination system, the offspring will always be female. An example is the "Little Fire Ant" (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments. Artificial cloning of organisms Artificial cloning of organisms may also be called reproductive cloning. First moves Hans Spemann, a German embryologist was awarded a Nobel Prize in Physiology or Medicine in 1935 for his discovery of the effect now known as embryonic induction, exercised by various parts of the embryo, that directs the development of groups of cells into particular tissues and organs. In 1928 he and his student, Otto Mangold, were the first to performsomaticcell nuclear transfer using amphibian embryos one of the first moves towards cloning. Methods Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg that has no nucleus. If the egg begins to divide normally it is transferred into the uterus of the surrogate mother. Such clones are not strictly identical since the somatic cells may contain mutations in their nuclear DNA. Additionally, the mitochondria in the cytoplasm also contains DNA and during SCNT this mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to death. Artificial embryo splitting or embryo twinning may also be used as a method of cloning, where an embryo is split in the maturation before embryo transfer. It is optimally performed at the 6- to 8-cell stage, where it can be used as an expansion of IVF to increase the number of available embryos. If both embryos are successful, it gives rise to monozygotic (identical) twins. Obtaining blastocysts A blastocyst is formed in the early stage of the development of an embryo. During cloning process, the blastocyst cells often are obtained by scientists 5 days after the egg has been divided.

Dolly the sheep

Dolly clone Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned from an adult cell. Dolly was formed by taking a cell from the udder of her biological mother. Her embryo was created by taking the cell and inserting it into a sheep ovum. The embryo was then placed inside a female sheep that went through a normal pregnancy. She was cloned at the Roslin Institute in Scotland and lived there from her birth in 1996 until her death in 2003 when she was six. Her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland. Dolly was publicly significant because the effort showed that genetic material from a specific adult cell, programmed to express only a distinct subset of its genes, can be reprogrammed to grow an entirely new organism. Before this demonstration, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg. However, this concept was not yet demonstrated in a mammalian system. Cloning Dolly the sheep had a low success rate per fertilized egg; she was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which 5

lived. Seventy calves have been created and one third of them died young;Prometea took 814 attempts. Notably, although the first clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell. There were early claims that Dolly the Sheep had pathologies resembling accelerated aging. Scientists speculated that Dolly's death in 2003 was related to the shortening of telomeres, DNA-protein complexes that protect the end of linear chromosomes. However, other researchers, including Ian Wilmut who led the team that successfully cloned Dolly, argue that Dolly's early death due to respiratory infection was unrelated to deficiencies with the cloning process. Species cloned The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Notable experiments include:

Tadpole: (1952) Robert Briggs and Thomas J. King had successfully cloned northern leopard frogs: thirty-five complete embryos and twenty-seven tadpoles from one-hundred and four successful nuclear transfers.

Carp: (1963) In China, embryologist Tong Dizhou produced the world's first cloned fish by inserting the DNA from a cell of a male carp into an egg from a female carp. He published the findings in a Chinese science journal.

Mice:

(1986)

mouse

was

successfully

cloned

from

an

early

embryonic

cell. Soviet scientists Chaylakhyan, Veprencev, Sviridova, and Nikitin had the mouse "Masha" cloned. Research was published in the magazine "Biofizika" volume II, issue 5 of 1987.

Sheep: Marked the first mammal being cloned (1984) from early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep from a somatic cell in 1996.

Rhesus Monkey: Tetra (January 2000) from embryo splitting Gaur: (2001) was the first endangered species cloned. Cattle: Alpha and Beta (males, 2001) and (2005) Brazil Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons Rat: Ralph, the first cloned rat (2003) Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-family clone. Horse: Prometea, a Haflinger female born 28 May 2003, was the first horse clone.

Dog: Snuppy, a male Afghan hound was the first cloned dog (2005). Wolf: Snuwolf and Snuwolffy, the first two cloned female wolves (2005). Water Buffalo: Samrupa was the first cloned water buffalo. It was born on February 6, 2009, at India's Karnal National Diary Research Institute but died five days later due to lung infection.

Pyrenean Ibex (2009) was the first "extinct" animal (while the species is not extinct, nor even endangered, no living examples of the Pyrenean subspecies had been known since 2000) to be cloned back to life; the clone lived for seven minutes before dying of lung defects.

Camel: (2009) Injaz, is the first cloned camel. Pashmina goat: (2012) Noori, is the first cloned pashmina goat. Scientists at the faculty of veterinary sciences and animal husbandry of Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir successfully cloned the first Pashmina goat (Noori) using the advanced reproductive techniques under the leadership of Riaz Ahmad Shah.[

Human cloning Human cloning is the creation of a genetically identical copy of an existing or previously existing human. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction. There are two commonly discussed types of human cloning: therapeutic cloning and reproductive cloning. Therapeutic cloning involves cloning adult cells for use in medicine and is an active area of research. Reproductive cloning would involve making cloned humans. A third type of cloning called replacement cloning is a theoretical possibility, and would be a combination of therapeutic and reproductive cloning. Replacement cloning would entail the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant. The various forms of human cloning are controversial. There have been numerous demands for all progress in the human cloning field to be halted. Most scientific, governmental and religious organizations oppose reproductive cloning. The American Association for the Advancement of Science (AAAS) and other scientific organizations have made public statements suggesting that human reproductive cloning be banned until safety issues are resolved. Serious ethical concerns have been raised by the future possibility of harvesting organs from clones. Some people have considered the idea of growing organs separately from a human organism - in doing this, a new organ supply could be established without the moral implications of harvesting them from humans. Research is also being done on the idea of growing organs that are

biologically acceptable to the human body inside of other organisms, such as pigs or cows, then transplanting them to humans, a form of xenotransplantation. The first hybrid human clone was created in November 1998, by Advanced Cell Technologies. It was created from a man's leg cell, and a cow's egg whos DNA was removed. It was destroyed after 12 days. Since a normal embryo implants at 14 days, Dr Robert Lanza, ACT's director of tissue engineering, told the Daily Mail newspaper that the embryo could not be seen as a person before 14 days. While making an embryo, which may have resulted in a complete human had it been allowed to come to term, according to ACT: "[ACT's] aim was 'therapeutic cloning' not 'reproductive cloning'" On January, 2008, Wood and Andrew French, Stemagen's chief scientific officer in California, announced that they successfully created the first 5 mature human embryos using DNA from adult skin cells, aiming to provide a source of viable embryonic stem cells. Dr. Samuel Wood and a colleague donated skin cells, and DNA from those cells was transferred to human eggs. It is not clear if the embryos produced would have been capable of further development, but Dr. Wood stated that if that were possible, using the technology for reproductive cloning would be both unethical and illegal. The 5 cloned embryos, created in Stemagen Corporation lab, in La Jolla, were destroyed. Ethical issues of cloning Because of recent technological advancements, the cloning of animals (and potentially humans) has been an issue. Many religious organizations oppose all forms of cloning. Judaism does not equate life with conception and, though some question the wisdom of cloning, Orthodox Judaism rabbis generally find no firm reason in Jewish law and ethics to object to cloning. From the standpoint of classical liberalism, concerns also exist regarding the protection of the identity of the individual and the right to protect one's genetic identity. Gregory Stock is a scientist and outspoken critic against restrictions on cloning research. The social implications of an artificial human production scheme were famously explored in Aldous Huxley's novel Brave New World. On December 28, 2006, the U.S. Food and Drug Administration (FDA) approved the consumption of meat and other products from cloned animals. Cloned-animal products were said to be virtually indistinguishable from the non-cloned animals. Furthermore, companies would not be required to provide labels informing the consumer that the meat comes from a cloned animal.

Critics have raised objections to the FDA's approval of cloned-animal products for human consumption, arguing that the FDA's research was inadequate, inappropriately limited, and of questionable scientific validity. Several consumer-advocate groups are working to encourage a tracking program that would allow consumers to become more aware of cloned-animal products within their food. Joseph Mendelson, legal director of the Center for Food Safety, said that cloned food still should be labeled since safety and ethical issues about it remain questionable. Carol Tucker Foreman, director of food policy at the Consumer Federation of America, stated that FDA does not consider the fact that the results of some studies revealed that cloned animals have increased rates of mortality and deformity at birth. Another concern is that the biotechnologies used on animals may someday be used on humans. Some people may be more open to the idea of cloning of animals because most western countries have passed legislation against cloning humans, yet only a few countries passed legislation against cloning animals. Possible Abnormalities due to Cloning Researchers have found several abnormalities in cloned organisms, particularly in mice. The cloned organism may be born normal and resemble its non-cloned counterpart, but majority of the time will express changes in its genome later on in life. The concern with cloning humans is that the changes in genomes may not only result in changes in appearance, but in psychological and personality changes as well. The theory behind this is that the biological blueprint of the genes is the same in cloned animals as it is in normal ones, but they are read and expressed differently. DNA arrays were used to prove this claim in the research lab of Professor Rudolf Jaenisch. Jaenisch studied placentas from cloned mice and found that one in every 25 genes was expressed abnormally. Results of these abnormally expressed genes in the cloned mice were premature death, pneumonia, liver failure and obesity. Cloning extinct and endangered species Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream of some scientists. The possible implications of this were dramatized in the best-selling novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. In real life, one of the most anticipated targets for cloning was once the Woolly Mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint RussoJapanese team is currently working toward this goal. And in January 2011, it was reported by Yomiuri Shimbun that a team of scientists headed by Akira Iritani of Kyoto University had built 9

upon research by Dr. Wakayama, saying that they will extract DNA from a mammoth carcass that had been preserved in a Russian laboratory and insert it into the egg cells of an African elephant in hopes of producing a mammoth embryo. The researchers said they hoped to produce a baby mammoth within six years. In 2001, a cow named Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after two days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean Ibex) were frozen in liquid nitrogen immediately after it died in 2000. Researchers are also considering cloning endangered species such as the giant panda and cheetah. The "Frozen Zoo" at the San Diego Zoo now stores frozen tissue from the world's rarest and most endangered species. In 2002, geneticists at the Australian Museum announced that they had replicated DNA of the Thylacine (Tasmanian Tiger), at the time extinct for about 65 years, using polymerase chain reaction However, on February 15, 2005 the museum announced that it was stopping the project after tests showed the specimens' DNA had been too badly degraded by the (ethanol) preservative. On 15 May 2005 it was announced that the Thylacine project would be revived, with new participation from researchers in New South Wales and Victoria. In January 2009, for the first time, an extinct animal, the Pyrenean ibex mentioned above was cloned, at the Centre of Food Technology and Research of Aragon, using the preserved DNA of the skin samples from 2001 and domestic goat egg-cells. (The ibex died shortly after birth due to physical defects in its lungs.) One of the continuing obstacles in the attempt to clone extinct species is the need for nearly perfect DNA. Cloning from a single specimen could not create a viable breeding population in sexually reproducing animals. Furthermore, even if males and females were to be cloned, the question would remain open whether they would be viable at all in the absence of parents that could teach or show them their natural behavior. Cloning endangered species is a highly ideological issue. Many conservation

biologists and environmentalists vehemently oppose cloning endangered speciesmainly because they think it may deter donations to help preserve natural habitat and wild animal populations. The "rule-of-thumb" in animal conservation is that, if it is still feasible to conserve habitat and viable wild populations, breeding in captivity should not be undertaken in isolation. In a 2006 review, David Ehrenfeld concluded that cloning in animal conservation is an experimental technology that, at its state in 2006, could not be expected to work except by pure chance and utterly failed a cost-benefit analysis Furthermore, he said, it is likely to siphon funds 10

from established and working projects and does not address any of the issues underlying animal extinction (such as habitat destruction, hunting or other overexploitation, and an impoverished gene pool). While cloning technologies are well-established and used on a regular basis in plant conservation, care must be taken to ensure genetic diversity. He concluded: Vertebrate cloning poses little risk to the environment, but it can consume scarce conservation resources, and its chances of success in preserving species seem poor. To date, the conservation benefits of transgenics and vertebrate cloning remain entirely theoretical, but many of the risks are known and documented. Conservation biologists should devote their research and energies to the established methods of conservation, none of which require transgenics or vertebrate cloning. On the 7 December 2011 it was announced that a team from the Siberian mammoth museum and Japan's Kinki University plan to clone a woolly mammoth from a well preserved sample of bone marrow found in August 2011. The team claim that the cloning could be complete within the next five years. Others have expressed doubt about the feasibility of the experiment. Scientists at the University of New South Wales announced in March 2013 that the very recently extinct gastric-brooding frog would be the subject of a cloning attempt to resurrect the species.

1. OVERVIEW OF METHODOLOGY FOR INSERTION OF GENES AND VECTORS The process of insertion of foreign genes and vectors is used to create recombinant DNA which can be inserted into a host for cloning. This would create multiple copies of the gene of interest. This method is a relatively new procedure which allows analysis of proteins and other parts of most organisms which had previously been very difficult to study due to the scarcity of the gene of interest. A genome is very large and the gene of interest is usually found only one or two times per cell. This new method allows the introduction of almost any type of genetic information into an organism. For example, a fragment of genome which contains the gene involved in the production of a toxin, can be removed from the bacteria from which it was found using restriction enzymes. The gene can be inserted into a cloning vector, forming recombinant DNA. The newly formed recombinant DNA can be inserted into the appropriate host, most likely E. coli, by the process of Transformation. The cells produced will contain the recombinant DNA which can be analyzed for possible

11

use in a vaccine. (All figures are fromwww.blc.arizona.edu/marty/181/181Lectures98/Lecture18_98.html )

FIGURE 1. Illustration of the procedure to form recombinant DNA.

FIGURE 2. Illustration of Transformation procedure.

12

2. OVERVIEW OF INSERTION OF GENE AND VECTOR PROCEDURES The procedure for insertion of foreign genes and vectors can be grouped into four categories based on the construction and insertion of recombinant DNA: DNA source, selected vector, restriction enzymes, transformation into host . The source of DNA used to construct the recombinant DNA, is usually a small part of a genome that contains the gene or sequence of interest. The gene or sequence of interest is usually of significant importance due to its unique function , whether it be the production of a protein, or capsule, or regulate gene expression. Once the DNA source is chosen, it must be isolated. This can be accomplished with the use of restriction endonucleases. The isolated DNA is then inserted into a vector. Vectors are used as a vehicle by which foreign DNA can be inserted into cells , so the recombinant DNA may be replicated. The most common type of vector is a plasmid. Plasmids are a small circular piece of extrachromosomal DNA found in bacterial cells, which are capable of replication independent of the host. Plasmids usually carry a few genes which give them a selective advantage, such as genes for antibiotic resistance.

13

FIGURE 3. Common cloning vector which contains restriction sites and genes for antibiotic resistance. Restriction endonucleases are used to cut the DNA and vector so they can be joined. Restriction endonucleases are bacterial enzymes that cleave DNA into fragments based on the recognition of specific nucleotide sequences. The enzymes are named after the bacterial species from which they were isolated. The DNA and vector to be joined are cut with the same restriction enzymes which can produce "sticky ends". DNA ligase is used to join the ends of the vector and DNA pieces. The newly formed recombinant DNA is inserted into a host, usually E.coli., by the process of transformation. As the cell divides, more cells are produced, each containing plasmid DNA which also exhibits antibiotic resistance. This process of making copies of foreign DNA is called molecular cloning. The cloned cells can be screened for by using antibiotic resistance markers.

3. PROS AND CONS Problems that may arise in the process of creating Recombinant DNA include; difficulty in location and isolation of the sequence of interest, finding the appropriate vector and successful transformation of the recombinant DNA into the host cell. The recombinant DNA must be made competent by treatment with a calcium chloride solution, but this does not insure that the procedure will work. One important concern with using this technique to produce recombinant DNA and inevitably cloned cells, is the use of the genetically engineered cells for biological or environmental purposes. Transgenic plants and animals could be created but the uncertainty of their impact on society may not permit their use until further research is conducted. This method however, has enabled the analysis of many proteins, enzymes and genes which previously had not been possible. this method has a wide variety of applications. It can be used in the medical field to study specific genes that might be linked to genetic disorders. It can be used in the creation of recombiant vaccines. This method can be applied in the agricultural field by creating transgenic plants which exhibit antibiotic resistance.

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Transformation (genetics)
From Wikipedia, the free encyclopedia

In this image, a gene from bacterial cell 1 is moved from bacterial cell 1 to bacterial cell 2. This process of bacterial cell 2 taking up new genetic material is called transformation.

Not to be confused with an unrelated process called malignant transformation which occurs in the progression of cancer. In molecular biology, transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be affected by artificial means in other cells. For transformation to happen, bacteriamust be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium). "Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA intoeukaryotic cells is often called "transfection".[1]

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Contents
[hide]

1 History 2 Methods and mechanisms

2.1 Bacteria

2.1.1 Natural transformation 2.1.2 Transformation, as an adaptation for DNA repair 2.1.3 Natural competence 2.1.4 Artificial competence

2.2 Yeast

o o

2.2.1 Transformation of spheroplasts 2.2.2 Transformation of intact yeast cells 2.2.3 Other yeast transformation methods

2.3 Plants 2.4 Animals

3 Practical aspects of transformation in molecular biology

3.1 Selection and screening in plasmid transformation

4 References 5 External links

History[edit]
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Transformation was first demonstrated in 1928 by British bacteriologist Frederick Griffith. Griffith discovered that a harmless strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation" (See Avery-MacLeod-McCarty experiment). The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development ofgenetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted.[2] It was originally thought that Escherichia coli, a commonly used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that E. coli may be induced to take up DNA from bacteriophage without the use of helper phage after treatment with calcium chloride solution.[3] Two years later in 1972, Stanley Cohen, Annie Chang and Leslie Hsu showed that CaCl
2 treatment is also effective for transformation of plasmid DNA.
[5] [4]

The method of transformation by Mandel and

Higa was later improved upon by Douglas Hanahan. The discovery of artificially induced competence in E. coli created an efficient and convenient procedure for transforming bacteria which allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely used laboratory procedure. Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed.[6] Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982.[7] In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid.[8] By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants.[9] Not all plant cells are susceptible to infection by A. tumefaciens so other methods were developed including electroporation and micro-injection.[10] Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.[11][12][13]

Methods and mechanisms[edit]

Bacteria[edit]
Bacterial transformation may be referred to as a stable genetic change brought about by the uptake of naked DNA (DNA without associated cells or proteins) and competencerefers to the state of being able to take up

17

exogenous DNA from the environment. There are two forms of transformation and competence: natural and artificial.

Natural transformation[edit]
Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be designed to carry out this process.[14][15] In general, transformation is a complex, energy requiring developmental process. In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome it must become competent, that is, enter a special physiological state. Competence development in Bacillus subtilis requires expression of about 40 genes.[16] The DNA integrated into the host chromosome is usually (but with rare exceptions) derived from another bacterium of the same species, and is thus homologous to the resident chromosome. In B. subtilis the length of the transferred DNA is greater than 1271 kb (more than 1 million bases). [17] The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb.[18] It appears that about 7-9% of the recipient cells take up an entire chromosome.[19] The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla) are known to undergo this process.[15] Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth. Transformation in Haemophilus influenzae occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase. [20] Transformation inStreptococcus mutans, as well as in many other streptococci, occurs at high cell density and is associated with biofilm formation.
[21]

Competence in B. subtilis is induced toward the end of

[22]

logarithmic growth, especially under conditions of amino acid limitation.

Transformation, as an adaptation for DNA repair[edit]

Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in Streptococcus pneumoniae by the DNA damaging agents mitomycin C (a DNA crosslinking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks).[23] In B. subtilis, transformation is increased by UV light, a DNA damaging agent.[24] In Helicobacter pylori, ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation[25] Using Legionella pneumophila, Charpentier et al.[26] tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents caused strong induction. These DNA damaging agents were mitomycin C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks [27]), bicyclomycin (causes single- and double-strand breaks[28]), and hydroxyurea (induces DNA base oxidation[29]).

18

UV light also induced competence in L. pneumophila. Charpentier et al. transformation probably evolved as a DNA damage response.

[26]

suggested that competence for

Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important DNA repair process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair process that is especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation.[30] To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using B. subtilis irradiated by UV light as the damaging agent (reviewed by Michod et al.[31] and Bernstein et al.[30]) The results of these experiments indicated that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular process responsible for repair was likely HRR. Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes (see Wikipedia articles Evolution of sexual reproduction; Meiosis.)

Natural competence[edit]
Main article: Natural competence About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria, in a process that is called horizontal gene transfer. Some species upon cell death release their DNA to be taken up by other cells, however transformation works best with DNA from closely related species. These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogeneous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.[14] Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase.[32] Only single-stranded

19

DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence however its role is uncertain.[33] The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake. [34]

Artificial competence[edit]

Schematic of bacterial transformation.

Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.[35] Typically the cells are incubated in a solution containing divalent cations (oftencalcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). However, the mechanism of the uptake of DNA via chemically induced competence in this calcium chloride transformation method is unclear. The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall. Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.

Yeast[edit]
Most species of yeast, including Saccharomyces cerevisiae, may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab. [36]

Transformation of spheroplasts[edit]

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Yeast cells may be treated with enzymes to degrade their cell walls, yielding spheroplasts. These cells are very fragile but take up foreign DNA at a high rate.[37]

Transformation of intact yeast cells[edit]

Exposing intact yeast cells to alkali cations such as those of cesium or lithium allows the cells to take up plasmid DNA.[38] Later protocols adapted this transformation method, using lithium acetate, polyethylene glycol, and single-stranded DNA. [39] In these protocols, the single-stranded DNA preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving it available for transformation.
[40]

Other yeast transformation methods[edit]

Enzymatic digestion,[41] electroporation,[42] or agitation with glass beads [43] may also be used to transform yeast cells.

Plants[edit]
A number of methods are available to transfer DNA into plant cells:

Agrobacterium mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid containing suspended Agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately, many plants are not transformable by this method.

Gene gun: Also referred to as particle bombardment, microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower than in Agrobacterium mediated transformation, but most plants can be transformed with this method.

Electroporation: make transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria.

Viral transformation (transduction): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus free and also free of the inserted gene.

Animals[edit]
21

Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.

Practical aspects of transformation in molecular biology[edit]

Further information: Transformation efficiency The discovery of artificially induced competence in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in E. coli. In order to be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome. The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as Transformation efficiency and is measured in colony forming unit (cfu) per g DNA used. A transformation efficiency of 1x108 cfu/g for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed. In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+ (in CaCl
2 solution) making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA,

and then briefly heat-shocked (e.g., at 42C for 30120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/g or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid.[44] Protocols however exist for making supercompetent cells that may yield a transformation efficiency of over 109.[45] The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA. The transformation efficiency using the CaCl
2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the

uptake of large plasmid DNA.[46] Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process.

Selection and screening in plasmid transformation[edit]

Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or have their growth arrested. Antibiotic resistance is the most commonly used marker for prokaryotes. The

22

transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow. In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the insert. Reporter genes can be used as markers, such as the lacZ gene which codes for -galactosidase used in blue-white screening. This method of screening relies on the principle of -complementation, where a fragment of the lacZgene (lacZ) in the plasmid can complement another mutant lacZ gene (lacZM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ- is transformed into a lacZM15 cells, they form a functional -galactosidase. The presence of an active galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligatedinto the plasmid vector, is located within the lacZ gene. Successful ligation therefore disrupts the lacZ gene, and no functional galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones. Other commonly used reporter genes are green fluorescent protein (GFP), which produces cells that glow green under blue light, and the enzyme luciferase, which catalyzes a reaction with luciferin to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.

Referen

23

Transfection
From Wikipedia, the free encyclopedia

Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells.[1] It may also refer to other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non-viral DNA transfer in bacteria, nonanimal eukaryotic cells and plant cells a distinctive sense of transformation refers to spontaneous genetic modifications (mutations to cancerous cells (carcinogenesis), or under stress (UV irradiation)).Transduction is often used to describe virus-mediated DNA transfer. The word transfection is a blend of trans- and infection. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material. Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Transfection can result in unexpected morphologies and abnormalities in target cells.
Contents
[hide]

1 Terminology 2 Methods

o o o o o

2.1 Chemical-based transfection 2.2 Non chemical methods 2.3 Particle-based methods 2.4 Viral methods 2.5 Other (and hybrid) methods

3 Stable and transient transfection 4 RNA transfection 5 See also 6 References 7 External links

Terminology[edit]

24

The meaning of the term has evolved.[2] The original meaning of transfection was "infection by transformation," i.e., introduction of DNA (or RNA) from a prokaryote-infecting virus orbacteriophage into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA.

Methods[edit]
There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials or biological particles (viruses) that are used as carriers.

Chemical-based transfection[edit]
Chemical-based transfection can be divided into several kinds: cyclodextrin,[3] polymers,[4] liposomes, or nanoparticles [5] (with or without chemical or viral functionalization. See below).

One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[6] (see also [7]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.

Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell.

A very efficient method is the inclusion of the DNA to be transfected in liposomes, i.e. small, membranebounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells, transfection is better achieved using cationic liposomes (or mixtures), because the cells are more sensitive. See lipofection for more details.

Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.

Non chemical methods[edit]

25

Electroporation (Gene electrotransfer) is a popular method, where transient increase in the permeability of cell membrane is achieved when the cells are exposed to short pulses of an intense electric field.

Similarly, transfection applying sonic forces to cells, referred as Sono-poration. Optical transfection is a method where a tiny (~1 m diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser. This technique was first described in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient transfection of normal rat kidney cells.[8] In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.

Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, or electroporation)are used in order to fuse the protoplast carrying the gene of interest with the target recipient cell. A major disadvantage of this method is that bacterial components are non-specifically introduced into the target cell as well.

Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is inserted into a cell. This approach can also be implemented with arrays of nanofibers that are introduced into large numbers of cells and intact tissue.

Hydrodynamic delivery In mice and rats, but to a lesser extent in larger animals, DNA most often in plasmids, including transposons, can be delivered to the liver using hydrodynamic injection that involves infusion of a relatively large volume in the blood in less than 10 seconds; nearly all of the DNA is expressed in the liver by this procedure.[9][10][11]

Particle-based methods[edit]

A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell'snucleus.

Magnetofection, or Magnet assisted transfection is a transfection method, which uses magnetic force to deliver DNA into target cells. Nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid particle complexes towards and into the target cells, where the cargo is released.[12][13][14]

26

Impalefection is carried out by impaling cells by elongated nanostructures and arrays of such nanostructures such as carbon nanofibers or silicon nanowires which have been functionalized with plasmid DNA.

Viral methods[edit]
DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and the cells are said to be transduced.

Other (and hybrid) methods[edit]

Other methods of transfection include nucleofection, heat shock.

Stable and transient transfection[edit]

For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded. Cell lines expressing the EpsteinBarr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution. [15] If it is desired that the transfected gene actually remain in the genome of the cell and its daughter cells, a stable transfection must occur. To accomplish this, a marker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have integrated the foreign genetic material into their genome. If the toxin is then added to the cell culture, only those few cells with the marker gene integrated into their genomes will be able toproliferate, while other cells will die. After applying this selective stress (selection pressure) for some time, only the cells with a stable transfection remain and can be cultivated further. A common agent for selecting stable transfection is Geneticin, also known as G418, which is a toxin that can be neutralized by the product of the neomycin resistance gene.

RNA transfection[edit]
Main article: RNA transfection RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. The latter application is referred as siRNA transfection or RNA silencing, and has become a major application in research (to replace the "knock-down" experiments, to study the expression of proteins, i.e. of Endothelin-1[16]) with potential applications in gene-therapy. A limitation of the silencing approach rely on the toxicity of the transfection for cells, and its suspected effect on the expression of other genes/proteins.

27

See also[edit]

Protofection Transformation Transduction Cationic liposome Nucleofection Magnet assisted transfection Impalefection

Electroporation
From Wikipedia, the free encyclopedia

Diagram of the major components of anelectroporator with cuvette loaded.

Dr.Eberhard Neumann - Electroporation Founder

28

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.[1] Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5 V to 1 V). This leads to the definition of an electric field magnitude threshold for electroporation (Eth). That is, only the cells within areas where EEth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation. [2] In molecular biology, the process of electroporation is often used for the transformation of bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also have cell walls which are different from the lipid membranes and are made ofpeptidoglycan and its derivatives. However, the walls are naturally porous and only act as stiff shells that protect bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, the plasmids can be transferred into the cell after electroporation. Several hundred volts across a distance of several millimeters are typically used in this process. Afterwards, the cells have to be handled carefully until they have had a chance to divide producing new cells that contain reproduced plasmids. This process is approximately ten times as effective as chemical transformation.[1][3] This procedure is also highly efficient for the introduction of foreign genes in tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process of introducing foreign DNAs into eukaryotic cells is known as transfection.

Contents
[hide]

1 Laboratory practice 2 Electroporators 3 Medical applications 4 Physical mechanism 5 References

Laboratory practice[edit]

29

Cuvettes for electroporation. These areplastic with aluminium electrodes and a blue lid. They hold a maximum of 400 l.
Electroporation is done with electroporators, appliances that create an electro-magnetic field in the cell solution. The cell suspensionis pipetted into a glass or plastic cuvette which has two aluminum electrodes on its sides. For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to electroporation it is mixed with theplasmid to be transformed. The mixture is pipetted into the cuvette, the voltage and capacitance are set, and the cuvette is inserted into the electroporator. Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in the cuvette or in aneppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more to allow recovery of the cells and expression of antibiotic resistance, followed by spreading on agar plates. The success of the elecroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Solutions with high salt concentrations might cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria. For a further detailed investigation of the process more attention should be paid to the output impedance of the porator device and theinput impedance of the cells suspension (e.g. salt content). As the process needs direct electrical contact between the electrodes and the suspension, and is inoperable with isolated electrodes, obviously the process involves certain electrolytic effects, due to small currents and not only fields.

Electroporators[edit]
Electroporators come in two types: hand-held and bench-tops. Benchtop electroporators are generally used as common lab equipment, residing atop a central bench or hood. Some units offer the possibility of electroporating multiple samples at the same time with special electrode assemblies that fit into multi-well cell culture dishes. Benchtop electroporators can also be set to different operating parameters depending on whether or not the cell has a cell wall. In contrast, there is a handheld electroporator available ("The Cloning Gun"). Separate units to electroporate either bacterial and mammalian cells are cordless, rechargeable and use disposable pipectrodes, which combine elements of both cuvettes and pipettes. Their operating parameters are preset to the optimal parameters for transforming either bacteria or mammalian cells. Both types of electoporators have been used on a wide range of cells - including E. coli (for transformation) and mammalian cells such as neurons, astrocytes, neuroglia, lymphocytes, monocytes, fibroblasts, epithelial and endothelial cells from humans, mice, rats and monkeys (for transfection).

30

Medical applications[edit]
A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within a narrow range while leaving neighboring cells unaffected, and thus represents a promising new treatment for cancer, heart disease and other disease states that require removal of tissue.[4] A recent technique called non-thermal irreversible electroporation (N-TIRE) has proven successful in treating many different types of tumors and other unwanted tissue. This procedure is done using small electrodes (about 1mm in diameter), placed either inside or surrounding the target tissue to apply short, repetitive bursts of electricity at a predetermined voltage and frequency. These bursts of electricity increase the resting transmembrane potential (TMP), so that nanopores form in the plasma membrane. When the electricity applied to the tissue is above the electric field threshold of the target tissue, the cells become permanently permeable from the formation of nanopores. As a result, the cells are unable to repair the damage and die due to a loss of homeostasis. [5] NTIRE is unique to other tumor ablation techniques in that it does not create thermal damage to the tissue around it. Contrastingly, reversible electroporation occurs when the electricity applied with the electrodes is below the electric field threshold of the target tissue. Because the electricity applied is below the cells threshold, it allows the cells to repair their phospho lipid bilayer and continue on with their normal cell functions. Reversible electroporation is typically done with treatments that involve getting a drug or gene (or other molecule that is not normally permeable to the cell membrane) into the cell. Not all tissue has the same electric field threshold; therefore careful calculations need to be made prior to a treatment to ensure safety and efficacy.[6] One major advantage of using N-TIRE is that, when done correctly according to careful calculations, it only affects the target tissue. Proteins, the extracellular matrix, and critical structures such as blood vessels and nerves are all unaffected and left healthy by this treatment. This allows for a quicker recovery, and facilitates a more rapid replacement of dead tumor cells with healthy cells.[7] The first successful treatment of malignant cutaneous tumors implanted in mice was completed in 2007 by a group of scientists who achieved complete tumor ablation in 12 out of 13 mice. They accomplished this by to sending 80 pulses of 100 microseconds at 0.3Hz with an electrical field magnitude of 2500 V/cm to treat the cutaneous tumors.[8] Before doing the procedure, scientists must carefully calculate exactly what needs to be done, and treat each patient on an individual case-by-case basis. To do this, imaging technology such as CT scans and MRIs are commonly u sed to create a 3D image of the tumor. From this information, they can approximate the volume of the tumor and decide on the best course of action including the insertion site of electrodes, the angle they are inserted in, the voltage needed, and more, using software technology. Often, a CT machine will be used to help with the placement of electrodes during the procedure, particularly when the electrodes are being used to treat tumors in the brain.[9] The entire procedure is very quick, typically taking about five minutes. The success rate of these procedures is high and is very promising for future treatment in humans. One disadvantage to using N-TIRE is that the electricity delivered from the electrodes can stimulate muscle cells to contract, which could have lethal consequences depending on the situation. Therefore, a paralytic agent must be used when performing the procedure. The paralytic agents that have been used in such research are successful; however, there is always some risk, albeit slight, when using anesthetics.

31

A more recent technique has been developed called high-frequency irreversible electroporation (H-FIRE). This technique uses electrodes to apply bipolar bursts of electricity at a high frequency, as opposed to unipolar bursts of electricity at a low frequency. This type of procedure has the same tumor ablation success as N-TIRE. However, it has one distinct advantage, H-FIRE does not cause muscle contraction in the patient and therefore there is no need for a paralytic agent.[10] Scientists have had high success rates in treating lab animals with many different kinds of tumors. Although scientists are not ready to routinely use irreversible electroporation to treat complicated tumors in humans, N-TIRE is more commonly used in humans to treat simple cutaneous or subcutaneous tumors.[11] In addition to treating cutaneous and subcutaneous tumors, there have been attempts at using this technology to treat prostate, lung, kidney and liver cancer in humans. However, scientists are still in the process of understanding this technology and its affect on animals and humans. One study done by Kenneth Thompson et al.[12] tested the safety of N-TIRE on humans suffering from lung, kidney or liver tumors. Of the 69 tumors treated, 49 of them achieved complete tumor ablation. The most successful treatment rate occurred in liver tumors. This study provides encouraging evidence for the future of the use of N-TIRE as a method of treating cancer in humans. Electroporation can also be used to help deliver drugs or genes into the cell by applying short and intense electric pulses that transiently permeabilize cell membrane, thus allowing transport of molecules otherwise not transported through a cellular membrane. This procedure is referred to as electrochemotherapy when the molecules to be transported is a chemotherapeutic agent or gene electrotransfer when the molecule to be transported is DNA.

Physical mechanism[edit]
Further information: Lipid bilayer mechanics Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core.[1] This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane. Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. In dielectric breakdown the barrier material is ionized, creating a conductive pathway. The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore which acts as the conductive pathway through the bilayer as it is filled with water.

32

Schematic showing the theoretical arrangement of lipids in a hydrophobic pore (top) and a hydrophilic pore (bottom).
Electroporation is a multi-step process with several distinct phases.[13] First, a short electrical pulse must be applied. Typical parameters would be 300-400 mV for < 1 ms across the membrane (note- the voltages used in cell experiments are typically much larger because they are being applied across large distances to the bulk solution so the resulting field across the actual membrane is only a small fraction of the applied bias). Upon application of this potential the membrane charges like a capacitor through the migration of ions from the surrounding solution. Once the critical field is achieved there is a rapid localized rearrangement in lipid morphology. The resulting structure is believed to be a pre-pore since it is not electrically conductive but leads rapidly to the creation of a conductive pore.[14] Evidence for the existence of such pre-pores comes mostly from the flickering of pores, which suggests a transition between conductive and insulating states. [15] It has been suggested that these pre-pores are small (~3 ) hydrophobic defects. If this theory is correct, then the transition to a conductive state could be explained by a rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic interface. Finally, these conductive pores can either heal, resealing the bilayer or expand, eventually rupturing it. The resultant fate depends on whether the critical defect size was exceeded[16] which in turn depends on the applied field, local mechanical stress and bilayer edge energy.

References[edit]

Microinjection
From Wikipedia, the free encyclopedia
Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the nuclear envelope. The desired contents are then injected into the desired sub-cellular compartment and the needle is removed. Microinjection is normally performed under a specialized optical microscope setup called a micromanipulator. The process is frequently used as a vector in genetic engineering and transgenics to insert genetic material into a single cell. Microinjection can also be used in the cloning of organisms, and in the study of cell biology and viruses.

Contents
[hide]

1 Subtypes

1.1 Pronuclear Injection

1.1.1 Pronuclear Injection in Mice

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1.1.2 Problems with Pronuclear Injection

1.2 Protoplast Injection

2 References 3 See Also

Subtypes[edit]
Pronuclear Injection[edit]
Pronuclear injection is a technique used to create transgenic organisms by injecting genetic material into nucleus of a fertilized oocyte. This technique is commonly used to study the role of genes using mouse models.

Pronuclear Injection in Mice[edit]

In order for pronuclear injection to be successful, the genetic material (typically linear DNA) must be injected while the genetic material from the oocyte and sperm are separate (i.e., the pronuclear phase).[1] In order to obtain these oocytes, mice are commonly superovulated using gonadotrophins.[2] Once plugging has occurred, oocytes are harvested from the mouse and injected with the genetic material. The oocyte is then implanted in the oviduct of a pseudopregnant animal.[1] While efficiency varies, 10-40% of mice born from these implanted oocytes may contain the injected construct.[2] Transgenic mice can then be bred to create transgenic lines.

Problems with Pronuclear Injection[edit]

The incorporation of injected DNA into the mouse genome cannot be tightly controlled. As a result, improper incorporation may occur. If DNA is incorporated at the 2-cell stage, some cells may express the DNA while others will not (so called mosaic mice). [2] In addition, if the injected DNA is incorporated in multiple sites, multiple copies of the DNA may be expressed leading to overexpression.[2] Even if only one copy of DNA is incorporated into the genome, if it is not incorporated into the germline, then it will not be passed tooffspring.[2]

Protoplast Injection[edit]
Microcapillary and microscopic devices can be used to deliver DNA into a protoplast.[3]

References[edit]

34

Gene gun
From Wikipedia, the free encyclopedia

35

PSD-1000/He Particle Delivery System

A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells withgenetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics. This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.

Contents
[hide]

1 Design 2 Application

o o

2.1 Plants 2.2 Humans and animals

3 References 4 Further reading 5 External links

Design[edit]
The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles. It was invented by John C Sanford, Ed Wolf and Nelson Allen at Cornell University,[1][2][3] and Ted Klein of DuPont, between 1983 and 1986. The original target was onions (chosen for their large cell size) and it was used to deliver particles coated with a marker gene.[4] Genetic transformation was then proven when the onion tissue expressed the gene. The earliest custom manufactured gene guns (fabricated by Nelson Allen) used a 22 caliber nail gun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel. A droplet of the tungsten powder and genetic material was placed on the bullet and shot down the barrel at a lexan "stopping" disk with a petri dish below. The bullet welded to the disk and the genetic information blasted into the sample in the dish with a doughnut effect (devastation in the middle, a ring of good transformation and little around the edge). The gun was connected to a vacuum pump and was under vacuum while firing. The early design was put into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Rd in Ithaca, NY, USA). Later the design was refined by removing the "surge tank" and changing to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to visually improve the machine for mass production to the scientific community. Biorad contracted with Dupont to manufacture and distribute the device. Improvements include the use of helium propellant and a multi-disk-collision delivery mechanism. Other heavy metals such as gold and silver are also used. Gold may be favored because it has better uniformity than tungsten and tungsten can be toxic to cells, but its use may be limited due to availability and cost.

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Applicationedit
Gene guns are so far mostly applied for plant cells. However, there is much potential use in animals and humans as well.

Plants[edit]
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated gold particle, whose DNA eventually migrates to and integrates into a plant chromosome. Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots. Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of total re-generation is called totipotency. The new plant that originated from a successfully shot cell may have new genetic (heritable) traits. The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its Ti plasmid to insert genetic information into plant cells. See transformation for different methods of transformation in different species.

Humans and animals[edit]

Gene guns have also been used to deliver DNA vaccines. The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG neurons, is also used as a pharmacological precursor in studying the effects of neurodegenerative diseases such as Alzheimer's Disease.

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The gene gun technique is also popularly used in an edible vaccine production technique, where the nano-gold particles coated with plant genetic material under the high vacuum pressurized chamber are transformed into suitable plant tissues. The gene gun has become a common tool for labeling subsets of cells in cultured tissue. In addition to being able to transfect cells with DNA plasmids coding for fluorescent proteins, the gene gun can be adapted to deliver a wide variety of vital dyes to cells. [5] Gene gun bombardment has also been used to transform C. elegans, as an alternative to microinjection.[6]

References[edit]

Somatic fusion
From Wikipedia, the free encyclopedia

This article may require cleanup to meet Wikipedia's quality standards. No cleanup reason has been specified. Please helpimprove this article if you can. (September 2010)

Fused protoplast (left) with chloroplasts (from a leaf cell) and coloured vacuole (from a petal).

Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between the different varieties of the same species (e.g. between

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non-flowering potato plants and flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale to produce Triticale). Uses of somatic fusion include making potato plants resistant to potato leaf roll disease. [1] Through somatic fusion, the crop potato plantSolanum tuberosum the yield of which is severely reduced by a viral disease transmitted on by the aphid vector is fused with the wild, non-tuber-bearing potato Solanum brevidens, which is resistant to the disease. The resulting hybrid has the chromosomes of both plants and is thus similar to polyploid plants. Somatic Hybridization was firstly introduced by Carlson[who?] in Nicotiana 'glauea'
Contents
[hide]

1 Process for plant cells 2 Applications in animal cells 3 Characteristics of Somatic Hybridization and Cybridization

3.1 Inter-specific and inter-generic fusion achievements

4 References

Process for plant cells[edit]

The somatic fusion process occurs in four steps:[2] 1. The removal of the cell wall of one cell of each type of plant using cellulase enzyme to produce a somatic cell called a protoplast 2. The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse together the nuclei. The resulting fused nucleus is calledheterokaryon. 3. The somatic hybrid cell then has its cell wall induced to form using hormones 4. The cells are then grown into calluses which then are further grown to plantlets and finally to a full plant, known as a somatic hybrid. Different from the procedure for seed plants describe above, fusion of moss protoplasts can be initiated without electric shock but by the use of polyethylene glycol (PEG). Further, moss protoplasts do not need phytohormones for regeneration, and they do not form a callus.[3] Instead, regenerating moss protoplasts behave like germinating moss spores.[4] Of further note sodium nitrate and calcium ion at high pH can be used, although results are variable depending on the organism.[5]

Applications in animal cells[edit]

Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are useful in a variety of ways, e.g.,

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(i) to study the control of cell division and gene expression, (ii) to investigate malignant transformations, (iii) to obtain viral replication, (iv) for gene or chromosome mapping and for (v) production of monoclonal antibodies by producing hybridoma (hybrid cells between an immortalised cell and an antibody producing lymphocyte), etc. Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse somatic cells. Generally, human fibrocytes or leucocytes are fused with mouse continuous cell lines. When human and mouse cells (or cells of any two mammalian species or of the same species) are mixed, spontaneous cell fusion occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of ultraviolet inactivated Sendai (parainfluenza) virus or polyethylene glycol (PEG). These agents adhere to the plasma membranes of cells and alter their properties in such a way that facilitates their fusion. Fusion of two cells produces a heterokaryon, i.e., a single hybrid cell with two nuclei, one from each of the cells entering fusion. Subsequently, the two nuclei also fuse to yield a hybrid cell with a single nucleus. A generalized scheme for somatic cell hybridization may be described as follows. Appropriate human and mouse cells are selected and mixed together in the presence of inactivated Sendai virus or PEG to promote cell fusion. After a period of time, the cells (a mixture of man, mouse and 'hybrid' cells) are plated on a selective medium, e.g., HAT medium, which allows the multiplication of hybrid cells only. Several clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic and appropriate biochemical analyses for the detection of enzyme/ protein/trait under investigation. An attempt is now made to correlate the presence and absence of the trait with the presence and absence of a human chromosome in the hybrid clones. If there is a perfect correlation between the presence and absence of a human chromosome and that of a trait in the hybrid clones, the gene governing the trait is taken to be located in the concerned chromosome. The HAT medium is one of the several selective media used for the selection of hybrid cells. This medium is supplemented with hypoxanthine, aminopterin and thymidine, hence the name HAT medium. Antimetabolite aminopterin blocks the cellular biosynthesis of purines and pyrimidines from simple sugars and amino acids.

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However, normal human and mouse cells can still multiply as they can utilize hypoxanthine and thymidine present in the medium through a salvage pathway, which ordinarily recycles the purines and pyrimidines produced from degradation of nucleic acids. Hypoxanthine is converted into guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), while thymidine is phosphorylated by thymidine kinase (TK); both HGPRT and TK are enzymes of the salvage pathway. On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+) enzymes can proliferate, while those deficient in these enzymes (HGPRr- and/or TK-) can not divide (since they cannot produce purines and pyrimidines due to the aminopterin present in the HAT medium). For using HAT medium as a selective agent, human cells used for fusion must be deficient for either the enzyme HGPRT or TK, while mouse cells must be deficient for the other enzyme of this pair. Thus, one may fuse HGPRT deficient human cells (designated as TK+ HGPRr-) with TK deficient mouse cells (denoted as TKHGPRT+). Their fusion products (hybrid cells) will be TK+ (due to the human gene) and HGPRT+ (due to the mouse gene) and will multiply on the HAT medium, while the man and mouse cells will fail to do so. Experiments with other selective media can be planned in a similar fashion.

Characteristics of Somatic Hybridization and Cybridization[edit]

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1. Somatic cell fusion appears to be the only means through which two different parental genomes can be recombined among plants that cannot reproduce sexually (asexual or sterile). 2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to produce fertile diploids and polyploids. 3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases somatic hybrids between two incompatible plants have also found application in industry oragriculture. 4. Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can be applied in plant breeding experiments.

Inter-specific and inter-generic fusion achievements[edit]

Cross Crossed with

Oat

Maize

Brassica sinensis

B. oleracea

Torrentia fourneri

T. bailloni

Brassica oleracea

B. campestris

Datura innoxia

Atropa belladonna

Nicotiana tabacum

N. glutinosa

Datura innoxia

D. candida

Arabidopsis thaliana Brassica campestris

Petunia hybrida

Vicia faba

Table: Reference #5 Note: The table only lists a few examples, there are many more crosses. The possibilities of this technology are great, however, not all species are easily put into protoplast culture.

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