There has been a long history of innovation and development of tools for gene discovery and genetic analysis in Drosophila melanogaster. This includes methods to induce mutations and to screen for those mutations that disrupt specific processes, methods to map mutations genetically and physically, and methods to clone and characterize genes at the molecular level.Modern genetics also requires techniques to do the reverse â€” to disrupt the functions of specific genes, the sequences of which are already known. This is the process referred to as reverse genetics.
Discovery of genes based on mutant phenotype is called as Forward genetics, now a days geneticists making use use of the reverse way ,where gene discovery starts with DNA sequence. Reverse genetics techniques are two types one is mostly like forward genetics screens using chemical mutagenesis or P element and other method is directed towards altering the function of gene of interest, targeted gene replacement and RNAi silencing falls into this category.
Today we will take a look at P-element mutagenesis ,as it happens to be most popular among tools of reverse genetics. Chemical mutagenesis is not the popular choice largely because of the lack of efficient and high-throughput methods for detecting mutations. P element mutagenesis a powerful tool to knock down a gene of interest. For gene disruption P element is modified in several ways to suit the experiment. Normal P element 2,907 bp includes a 31-bp inverted repeat at both ends and transposase gene ( required for mobilisation of P element, the transposon) has four exons.
P element needs only few bases including the inverted repeats to do its normal function. Making use of this property and the fact that laboratory strains of Drosophila are devoid of P elements, additionally these insertions are completely stable , makes p- element a great tool for generating mutations.Transposition can be induced at will by expressing transposase (by crossing to a fly harboring transposase source)This feature has allowed P-elements to become widely used in mutagenesis, gene tagging and transformation.
There are many P elements available in stock centers which are mapped, only thing one need to do is order the stock of P element sitting near to your gene of interest ( as P-elements hops in local regions) and provide transposase source for hoping into the gene so that it disrupts its coding region or one can order P elements already sitting in the gene and carry out imprecise excisions.
Imprecise excisions are very easy way of creating mutation in the gene. A marker gene, usually the white (w+) eye-color gene, is included so that integration and excision can be followed phenotypically. When a fly has this p element the eye color of fly looks red and when it has excised out of genome eye of fly becomes white.These are the flies of interest and possibly the mutation for the gene. Once the p element has taken the gene region along with it, its difficult for the p element to insert in genome again as it alters the inverted repeat sequence. One can replace one p element by another p element ( Lacz p element to Gal4 containing P element) with this technique.
Thomas Hummel and Christian KlÃ¤mbt
Drosophila , 97-117, DOI 10.1007/978-1-59745-583-1
Conversion of lacZ Enhancer Trap Lines to GAL4 Lines Using Targeted Transposition in Drosophila melanogaster
Katharine J. Seppa and Vanessa J. Auld
Genetics, Vol. 151, 1093-1101, March 1999
A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila.
de Navas L, Foronda D, Suzanne M, SÃ¡nchez-Herrero E.
Mech Dev. 2006 Nov;123(11):860-7.