Reverse and Forward genetics

There are two main kinds of genetic screens that are used to identify gene function in a high throughput
fashion


In reverse genetic screens, a large number of genes (or gene products) is
systematically inhibited one by one.

This can be accomplished many ways, for example by deleting genes using
homologous recombination or by selectively reducing messenger RNA
abundance.

Then, one or more phenotypes of interest are measured.

The main challenge of this approach is that for some organisms it is difficult to
disrupt large numbers of genes (such as tens of thousands) in a systematic
fashion.


It can also be challenging to discern the phenotypic consequences for a gene
that is disrupted.

As an example of reverse genetics, Thomas Sudhoff and colleagues targeted
the deletion of mouse syntaxin binding protein 1 (Stxb1; also called Munc18-1
or N-sec1), a gene encoding a nerve terminal protein (Verhage et al., 2000).

The phenotype was lethality at the time of birth, with neurons unable to secrete
neurotransmitter.

Remarkably, brain development appeared normal up to the time of death.

This targeted deletion allowed the dissection of the functional role of this gene.














In forward genetic screens, one begins with a defined
phenotype of interest, such as the ability of plants to grow in the
presence of a drug, or the ability of neurons to extend axons to
appropriate targets in the mammalian nervous system, or the
ability of a eukaryotic cell to transport cargo.

An experimental intervention is made, such as administering a
chemical mutagen or radiation to cells (or to an organism). This
results in the creation of mutants.

The phenotype of interest is observed in rare representatives
among a large collection of mutants

If individuals need to be assayed for the phenotype one at a time
(as part of a screen) this can be extremely laborious

If a specific selective condition can be defined in which only the
desired mutant grows (a selection) the process is greatly
facilitated.

A second challenge of forward genetics approaches is to then
identify the responsible gene(s) using mapping and sequencing
strategies.



The Yeast Saccharomyces cerevisiae
The budding yeast S. cerevisiae is the best characterized organism among the eukaroytes.
This single-celled fungus was the first eukaryote to have its genome sequenced.

Its 13 megabase genome encodes about 6000 proteins - The Saccharomyces Genome Database (SGD)

6608 annotated genes, including ~4600 that are verified, ~1100 that are uncharacterized (likely to be functional based
on conservation across speciesbut not experimentally validated), and ~800 dubious (open reading frames that are
neither well conserved nor validated).

Approximately 4200 gene products have been annotated to the root gene ontology terms (molecular function, biological
process, cellular component

To Learn SGD, performa search with a typical query, SEC1 .


The Plant Arabidopsis thaliana


The thale cress Arabidopsis thaliana was the first plant to have its genome sequenced (and the third finished eukaryotic
genome sequence).

It has served as a model for eukaryotic functional genomics projects.

The principal website, the Arabidopsis Information Resource (TAIR), centralizes a vast amount of information about its
genome (Swarbreck et al., 2008).

As an example of a gene search at TAIR, a query for Arabidopsis SEC1A(RefSeq accessionNP_563643; locus
tagAt1g02010) reveals information about its chromosomal location and available mutants


The Nematode Caenorhabditis elegans

Among the metazoans (animals), the soil-dwelling nematode Caenorhabditis elegans is a key model organism. This was
the first multicellular animal to have its genome sequenced.

This roundworm, like fruitflies and humans, is capable of complex behaviors, but its body is simple and all 959 somatic
cells in its body have been mapped, including their lineages throughout development. Wormbase is the main
on-line information repository.

The Fruitfly Drosophila melanogaster


The fruitfly Drosophila melanogaster, another metazoan invertebrate, has long served as a model for genetics.

Early studies of Drosophila resulted in the descriptions of the nature of the gene, as well as linkage and recombination,
producing gene maps a century ago.

The recent sequencing of 12 species of the Drosophila genus is already providing unprecedented insight
intomechanisms of genome evolution.

The central Drosophila database, FlyBase, combines molecular and genetic data on the Drosophilidae

A strength of Drosophila as a model organism is that genomic changes can be induced with extreme precision, from
single nucleotide changes to introducing large-scale chromosomal deletions, duplications, inversions,
or other modifications.

At the same time, it is a multicellular animal that features a complex body plan. Currently, loss of function mutations
have been introduced into all of its ~14,000 genes, and over half of these have an identifiable phenotype.








The Zebrafish Danio rerio


Although the lineages leading to modern fish and humans diverged approximately 450 million years ago, both are
vertebrate species, and orthologs are identifiable for the great majority of their protein-coding genes (with an average of
about 80% amino acid identity between orthologs).

The first four fish genomes to be sequenced were the pufferfish Takifugu rubripes and Tetraodon nigroviridis,
themedaka Oryzias latipes, and the zebrafish Danio rerio. Of these, the zebrafish has emerged as an important
model organism for functional genomics.

It is a small tropical freshwater fish having a genome size of 1.8 billion base pairs (Gb) organized into 25
chromsomes. For functional genomics studies, the zebrafish has served as a model for understanding both normal and
abnormal development. Mutations in large numbers of human disease gene orthologs have been generated and
characterized

Some of the advantages of zebrafish as a model organism include the following

Its generation time is short, especially for a vertebrate.

It produces large numbers of progeny.

The developing embryo is transparent. Thus, for example, if a transgene is inserted into the genome with a
promoter that drives the expression of green fluorescent protein (GFP), it is possible to see this expression from
the outside of each animals body.

It is a vertebrate and thus a close model for human disease.

Its genome is well annotated.

The vertebrate genome annotation (Vega) database at the Sanger Institute focuses on high quality manual
annotation
with a particular focus on just three genomes: human, mouse, and zebrafish

The principal zebrafish website is the Zebrafish Information Network


The Mouse Mus musculus


The rodents diverged from the primate lineage relatively recently (80 million years ago) and share almost all of their
genes with humans.

The mouse Mus musculus is one of the most important model organisms for the study of human gene function
because of the close structural and functional relationship between the two genomes,combined with a relatively short
generational span, and powerful tools have been developed to manipulate its genome. About 9000 mouse genes have
been knocked out.


There are currently three major mouse functional genomics initiatives (International Mouse Knockout Consortium,
2007): the Knockout Mouse Project (KOMP), The European Conditional Mouse Mutagenesis Program (EUCOMM),
and the North American Conditional Mouse Mutagenesis Project (NorCOMM).
TheMouse Genome Informatics (MGI) Database is the principal website for mouse genomics information