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International Journal of Morphology

Version On-line ISSN 0717-9502

CLONATION, PRODUCTION OF CHIMERAS AND PLURIPOTENCIAL


CELLS
**
Unit de Biologie du Dveloppement et Biotechnologie, INRA, Francia

ABSTRACT: Biology of Development and Biotechnology have advanced enormously in


recent years. Studies that are currently being carried out with new molecular techniques,
lead us to a new era in the diagnosis and treatment of many congenital diseases. At the
moment, it has been possible to apply the knowledge derived from it, with incipient
success, in the productive aspects and also in the therapy of different diseases of Man and
other animals and, finally, there are important perspectives in the protection of endangered
species. In this Review, we discuss some of the most interesting technical aspects of
cloning, production of chimeras and obtaining pluripotent cells, as well as objectives and
projections in the veterinary field and animal and human health.

KEY WORDS: 1. Cloning; 2. Chimeras; 3. Pluripotential cells.

Studies that are currently underway and using new molecular techniques and highly
sophisticated equipment, lead us to a new era in the diagnosis and treatment of many
congenital diseases. It has now been possible to apply the knowledge derived from it, with
incipient success, in the productive aspects and also in the therapy of different human and
other animal diseases and, finally, there are important perspectives in the protection of
threatened species. Next, we will analyze some aspects that have been permanent news
in recent years, such as cloning, production of chimeras, and obtaining stem cells (stem
cells).

Cloning: The techniques used for cloning can be of two types: 1) the one practiced by
transfer of nuclei, where individuals are generated that are equal copies to the progenitor,
like the dolly sheep and 2) the technique realized by gemelacin, starting from a Fertilized
ovum (in the traditional way), is split, producing two or more embryos. In this case, the
genetic constitution of the offspring is different from that of the parents, but is exactly the
same between them (Esponda, 2000).

Clonic mammals created by a nucleus transplant. We will refer to cloning in animals,


especially Dolly the sheep. This cloning technique for reproductive purposes should not be
used in humans. The rights of Man not to be genetically programmed and to be genetically
unique and unrepeatable constitute a serious and serious ethical objection against
reproductive cloning by somatic cell nucleus transfer. In 1997, Ian Wilmuth's group at the
Roslin Institute in Edinburgh achieved a cloned mammal through a core transplant. (No
male intervention). Dolly is the first case of a clonic mammal originated by a nonsexual
cell, and was created to obtain a replicate of a transgenic sheep that did not reproduce.
This was done as follows:
1) From a white headed sheep cells of a mammary gland were obtained, which were
maintained in a culture medium. From these cells the nuclei were extracted.

2) From sheep with black heads obtained eggs, which were removed by
micromanipulation the nucleus, being therefore exclusively constituted by its cytoplasm
and covered by the zona pellucida.

3) Nuclei extracted from mammary cells (from a white-headed sheep) were injected into
the cytoplasm of the eggs (from a black-headed sheep).

4) Oocytes containing the nucleus of the mammary cell were cultured for six days, and
those with embryonic development (up to blastocyst stage) were transferred to the uterus
of black headed sheep.

Thus was born Dolly, who possesses only the genetic information given by the nucleus of
the mammary cell and whose sex could only be female and the color of her head was
white (Fig. 1).

Pedro Esponda, in his entertaining book called "Beings of the Future" says that 277 eggs
were used that were injected with nuclei. Of these, 29 reached the embryonic state of
blastocyst and only one sheep was born that was Dolly. Therefore, the success of the
experiment was remarkably low (0.3%).

In synthesis, it is possible to affirm that three elements are indispensable: 1) a nucleus that
contains the complete hereditary information, 2) the cytoplasm of an ovule that must
correspond in principle to the same species as the transplanted nucleus and 3) a mother
Receptor that will nest in your uterus to the embryo. With respect to the value of the
species that nestles the embryo, it should be noted that there are cases where the uterus
of one species can nest a specimen of another species, such as a mare that served as a
host mother to a zebra, or The case of pandas that can develop in uterus of females of
other species of bears.

What seems irreplaceable is the cytoplasm of the ovule, whose composition is very
complex in structures and molecules, being able to organize everything so that the
embryonic divisions take place. The cytoplasm of the egg shows an unprecedented ability
to transform the gene expression of a nucleus already programmed by nature (like that of
cells already differentiated as the mammary gland). Such is the role of this ovular
cytoplasm, which is capable of causing these transplanted nuclei to be dedifferentiated,
reprogrammed and embryogenesis. Probably, the cytoplasm of the egg has molecules that
take part in this mechanism, but at this moment we do not know exactly which molecules it
is dealing with. The French group led by Xavier Vignon works on elucidating the molecular
mechanisms that allow the return of an already differentiated cell nucleus to a nucleus that
is totipotent.

Cloning offers important opportunities for the genomic preservation of animals at risk of
Animals at risk of extinction, but their efficiency is reduced. It has been considered that
one of the factors related to this low efficiency is the origin of the nucleus donor cells. Due
to this, in our group we performed an immunohistochemical study of cells obtained from
skin biopsies of adult bovines and neonates, after being cultured "in vitro" for periods of
one to three weeks.
REFERENCES

Brstle, O. et al. Embryonic stem cell-derived glial precursors: A source of myelinating


transplant. Science, 285: 754-6, 1999. [Links]

Sponda, P. Beings of the future "From in vitro fertilization to the clones and transgenics"
Mundo Vivo, 2000. [Links]

Dowsing, A.T .; Gougoulidis, T .; Dowsing, B. J .; Draber, P. & Trounson, A. O. The Stage-


Specific Expression of TEC-1, -2, -3, and -4 Antigens on Bovine preimplantation embryos.
Mol. Rep. Dev., 49: 19-28, 1998. [Links]

Geahart, J. New potential for human embryonic stem cells. Science, 282: 1061-2, 1998.
[Links]

Gilbert, S. Development Biology, Sinauer Associates. Sunderland, Massachusetts. 2000.


[Links]

Mc Donald, et al. Transplanted embryonic stem cells survive, differentiate, and promote
recovery in injured rat spinal cord. Nature Med., 5: 1410-2, 1999. [Links]

Montiel, E .; Guillomot, M .; Rojas, M .; Bustos-Obregn, E. & Flechon, J. Primordial germ


cell characterization by immunohistochemistry of vasa-homologue protein in
preimplantational rabbit embryos. Int J. Dev. Biol., 48: 141-2, 2001. [Links]

Rojas, M.A .; Vignon, X .; Montenegro, M.A .; Del Sol, M .; BustosObregn, E. & Flchon,
J. Characterization isolation and culture of primordial rabbit germ cells. Rev. Chil. Anat. 19
(2): 213-20, 2001. [Links]

Rojas, M.A .; Guillomot, M .; Montiel, E .; Venegas, F. & Bustos-Obregn, E. Specific


markers for the recognition of primordial germinal cells in the different stages of their
evolution. XIII Veterinary Congress, Valdivia, 2004. [Links]

Thomson, J.A. et al. Embryonic stem cells line derived from human blastocyst. Science
282: 1145-7, 1998. [Links]
Revista Internacional de Morfologa

Versin en lnea ISSN 0717-9502

CLONACIN, PRODUCCIN DE CHIMERAS Y CLULAS PLURIPOTENCIALES

** Unidad de Biologa del Desarrollo y Biotecnologa, INRA, Francia

RESUMEN: La Biologa del Desarrollo y la Biotecnologa ha avanzado enormemente en


los ltimos aos. Los estudios que se estn llevando a cabo actualmente con nuevas
tcnicas moleculares, nos llevan a una nueva era en el diagnstico y tratamiento de
muchas enfermedades congnitas. Actualmente, se ha podido aplicar el conocimiento
derivado de l, con incipiente xito, en los aspectos productivos y tambin en la terapia de
diferentes enfermedades del Hombre y otros animales y, finalmente, hay importantes
perspectivas en la proteccin de especies en peligro de extincin especies. En esta
Revista se discuten algunos de los aspectos tcnicos ms interesantes de la clonacin,
produccin de quimeras y obtencin de clulas pluripotentes, as como objetivos y
proyecciones en el campo veterinario y en salud animal y humana.

PALABRAS CLAVE: 1. Clonacin; 2. Quimeras; 3. Clulas pluripotenciales.

Los estudios que se estn llevando a cabo y utilizando nuevas tcnicas moleculares y
equipos altamente sofisticados, nos llevan a una nueva era en el diagnstico y tratamiento
de muchas enfermedades congnitas. Ahora es posible aplicar los conocimientos que se
derivan de l, con un xito incipiente, en los aspectos productivos y tambin en la terapia
de diferentes enfermedades humanas y de otros animales y, finalmente, hay perspectivas
importantes en la proteccin de especies amenazadas. A continuacin, analizaremos
algunos aspectos que han sido novedades permanentes en los ltimos aos, como la
clonacin, la produccin de quimeras y la obtencin de clulas madre.

Clonacin: Las tcnicas utilizadas para la clonacin pueden ser de dos tipos: 1) la
practicada por transferencia de ncleos, donde se generan individuos que son ejemplares
iguales al progenitor, como el ovino dolly y 2) la tcnica realizada por gemelacin, a partir
de Un vulo fertilizado (en la forma tradicional), se divide, produciendo dos o ms
embriones. En este caso, la constitucin gentica de la descendencia es diferente de la
de los padres, pero es exactamente la misma entre ellos (Esponda, 2000).

Mamferos clnicos creados por un trasplante de ncleo. Nos referiremos a la clonacin


en animales, especialmente Dolly la oveja. Esta tcnica de clonacin con fines
reproductivos no debe utilizarse en seres humanos. Los derechos del hombre a no ser
genticamente programados ya ser genticamente nicos e irrepetibles constituyen una
seria y seria objecin tica contra la reproduccin clonada mediante la transferencia de
ncleos de clulas somticas. En 1997, el grupo de Ian Wilmuth en el Instituto Roslin en
Edimburgo logr un mamfero clonado a travs de un trasplante de ncleo. (Ninguna
intervencin masculina). Dolly es el primer caso de un mamfero clnico originado por una
clula no sexual, y fue creado para obtener una rplica de una oveja transgnica que no
se reprodujo. Esto se hizo de la siguiente manera:
1) Se obtuvieron clulas de oveja de cabeza blanca de una glndula mamaria, que se
mantuvieron en un medio de cultivo. De estas clulas se extrajeron los ncleos.

2) De ovejas con cabezas negras se obtuvieron huevos, los cuales fueron eliminados por
micromanipulacin del ncleo, quedando por lo tanto exclusivamente constituidos por su
citoplasma y cubiertos por la zona pelcida.

3) Los ncleos extrados de las clulas mamarias (de una oveja de cabeza blanca) se
inyectaron en el citoplasma de los huevos (de una oveja de cabeza negra).

4) Los ovocitos que contienen el ncleo de la clula mamaria se cultivaron durante seis
das, y los que tenan desarrollo embrionario (hasta la etapa de blastocisto) se
transfirieron al tero de ovejas de cabeza negra.

As naci Dolly, que posee slo la informacin gentica dada por el ncleo de la clula
mamaria y cuyo sexo slo poda ser femenino y el color de su cabeza era blanco (Fig. 1).

Pedro Esponda, en su divertido libro titulado "Seres del Futuro", dice que se usaron 277
huevos que fueron inyectados con ncleos. De stos, 29 alcanzaron el estado
embrionario del blastocisto y naci solamente una oveja que era Dolly. Por lo tanto, el
xito del experimento fue notablemente bajo (0,3%).

En sntesis, es posible afirmar que son indispensables tres elementos: 1) un ncleo que
contiene la informacin hereditaria completa; 2) el citoplasma de un vulo que debe
corresponder en principio a la misma especie que el ncleo trasplantado; y 3) una madre
Receptor que anidar en su tero al embrin. Con respecto al valor de la especie que
anida al embrin, cabe sealar que hay casos en los que el tero de una especie puede
anidar un espcimen de otra especie, como una yegua que sirvi de madre husped a
una cebra, o El caso de los pandas que pueden desarrollarse en el tero de hembras de
otras especies de osos.

Lo que parece irremplazable es el citoplasma del vulo, cuya composicin es muy


compleja en estructuras y molculas, pudiendo organizar todo para que las divisiones
embrionarias tengan lugar. El citoplasma del huevo muestra una capacidad sin
precedentes para transformar la expresin gnica de un ncleo ya programa.
REFERENCIAS

Brstle, O. et al. Precursores glticos derivados de clulas madre embrionarias: Una


fuente de trasplante mielinizante. Science, 285: 754-6, 1999. [Enlaces]

Sponda, P. Seres del futuro "De la fecundacin in vitro a los clones y transgnicos" Mundo
Vivo, 2000. [Links]

Dowsing, A.T .; Gougoulidis, T.; Dowsing, B.J.; Draber, P. & Trounson, A. O. La Expresin
Especfica Especfica de los Antgenos TEC-1, -2, -3 y -4 en embriones de
preimplantacin bovina. Mol. Rep. Dev., 49: 19-28, 1998. [Enlaces]

Geahart, J. Nuevo potencial para las clulas madre embrionarias humanas. Science, 282:
1061-2, 1998. [Enlaces]

Gilbert, S. Biologa del Desarrollo, Sinauer Associates. Sunderland, Massachusetts. 2000.


[Enlaces]

Mc Donald, et al. Las clulas madre embrionarias trasplantadas sobreviven, se diferencian


y promueven la recuperacin en la mdula espinal daada de la rata. Nature Med., 5:
1410-2, 1999. [Enlaces]
Montiel, E.; Guillomot, M .; Rojas, M .; Bustos-Obregn, E. y Flechon, J. Caracterizacin
primordial de clulas germinales por inmunohistoqumica de la protena vasa-homloga en
embriones preimplantables de conejo. Int J. Dev. Biol., 48: 141 - 2, 2001. [Links]

Rojas, M.A .; Vignon, X.; Montenegro, M.A .; Del Sol, M.; BustosObregn, E. & Flchon, J.
Caracterizacin aislamiento y cultivo de clulas germinales primordiales de conejo. Rev.
Chil. Anat. 19 (2): 213-20, 2001. [Enlaces]

Rojas, M.A .; Guillomot, M .; Montiel, E.; Venegas, F. & Bustos-Obregn, E. Marcadores


especficos para el reconocimiento de las clulas germinales primordiales en las
diferentes etapas de su evolucin. XIII Congreso Veterinario, Valdivia, 2004. [Links]

Thomson, J.A. Et al. Lnea de clulas madre embrionarias derivadas del blastocisto
humano. Science 282: 1145 - 7, 1998. [Enlaces]

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