Question

Explain what forward genetics and reverse genetics are ? Assume you have known the function of the gene A via genetic analysis, design some experiments using both forward and reverse genetics approaches to identify the gene X that may work in the same pathway as the gene A .

Answer 1

1. Forward (classical) genetics – mutant phenotype -> gene

Forward genetics (or a forward genetic screen) is an approach used to identify genes (or set of genes) responsible for a particular phenotype of an organism.

Forward saturation genetics – treat organism (bacteria, C. elegans, Drosophila, Arabidopsis, etc.), with a mutagen, then screen offspring for particular phenotypes of interest. Examples: inability of bacteria to grow on certain sugars, problems in fly embryonic development, plants lacking a response to light. The goal is to find all of the genes involved in a trait. This approach is known as a “genetic screen”. Here, whatever the function of X gene is that will be lost. Mutagens:

a) X-rays – cause breaks in double-stranded DNA, resulting in large deletions of pieces of chromosome or chromosomal re-arrangements. These mutations are typically easy to map by cytological examination of chromosomes, but are often not limited to single genes. Not good for fine-scale mutagenesis.

b) Chemical – for example, the chemical ethylmethanesulfonate (EMS) causes point mutations, which are changes at a single nucleotide position. Mutations may be nonsense (introduce a premature stop codon) or mis-sense (cause an amino acid replacement). They may also be in non-coding sequence, affecting splicing signals or regulatory elements that control gene expression. This approach allows for many different mutations within gene regions, but these are difficult to map.

c) Insertional (transposon) mutagenesis – Transposable elements (TEs) containing a marker gene(s) are mobilized in the genome. The TE can insert within a coding region and disrupt the amino acid sequence or it may insert into neighboring non-coding DNA and affect intron splicing or gene expression. The major advantage is that the TE insertion can easily be mapped and the region of genome cloned.

2. Reverse genetics – gene -> mutant phenotype

Reverse genetics (or a reverse genetic screen), on the other hand, analyzes the phenotype of an organism following the disruption of a known gene.

In short, forward genetics starts with a phenotype and moves towards identifying the gene(s) responsible, whereas reverse genetics starts with a known gene and assays the effect of its disruption by analyzing the resultant phenotypes. Both forward and reverse genetic screens aim to determine gene function.

a) Large-scale random mutagenesis and screening – use forward mutagenesis as above (for example EMS), except instead of screening for a particular phenotype, screen your gene of interest for nucleotide changes. This typically requires that you screen 1000’s or 10,000’s of individuals. This is done by performing PCR on your gene of interest

b) Homologous recombination (HR) – works well in bacteria, yeast, mice (and some other mammals). It does not work well in Drosophila, although a complex experimental approach has been developed for this. HR has been used to knockout every predicted gene in yeast. It is possible to buy a set of about 6,000 yeast strains, each with a different gene knocked-out. Many mouse genes have been knocked out by this method. It has also been used to knockout a pig enzyme that links sugars into a form recognized as an antigen by the human body, with the long-term goal of engineering pig organs to be used for human transplants.

c) Transposable element excision – especially useful in Drosophila, where the Berkeley Drosophila Genome Project (BDGP) has a large collection of fly lines, each with a marked TE inserted at a unique chromosomal location. When a source of transposase is introduced, the TE will excise with some low frequency, resulting in a loss of the marker gene. Often the TE excision will also result in a deletion of the flanking DNA. Thus, if you have a TE insertion near your gene of interest, you may try to knockout your gene by excising the nearby TE.