How to identify rosy marker in Drosophila ?
Generation of transgenic Drosophila is possible by microinjection plasmid DNA containing the gene of interest into pole cells (located at one end of embryo) of pre blastoderm embryo. During this particular stage of embryogenesis nuclei are not surrounded by membrane (syncytium stage) and this makes many nuclei accessible for uptake of injected plasmid.
Drosophila transgenesis mainly relies on P-element which was for the first time used by Gerry Rubin and Alan Spradling in 1982.The use of p element for transformation was a major breakthrough in the germline transgenesis in Drosophila. However transgenesis in Fruit fly Drosophila melanogaster has come a long way from the days of landmark discovery and today its possible to insert the gene of your interest at any locus in the genome.
Unlike other animals, injected plasmid doesnot integrate into genome directly without any external help. Hence its important to depend on natural integration system that depends on transposable elements and P-elements happen to be most popular vehicles for plasmid integration in Drosophila.
In order to make transgenic flies first thing need to be done is to clone the gene which needs to be over expressed into modified P-element. These modified P-elements will contain one genetic marker ,which will eventually help in identifying transformants and non-transformants.
The first marker used for transformation was wild type rosy gene ry+,which encodes Xanthine dehydogenase enzyme.
This enzyme performs a vital reaction during synthesis of eye pigments. Wild type flies have red colour eyes ,whereas flies lacking rosy gene displays dark crimson to Brown eyes. So during transgenesis , DNA is injected in rosy mutant embryos and subsequently mated also with flies lacking rosy gene. When the injected plasmid containing rosy+ marker gets integrated into chromosome leads to a fly with wild type red eye colour. So thing to remember while using rosy gene is that one needs to be in white + background in order to see rosy gene.
The major advantage of using rosy gene is that even 1% of rosy gene expression is sufficient to produce ry+ eye colour. This helps in recovering transformants when the P-element is inserted into a location where it is transcribed less. However ry marker has some disadvantages like being big ~7kb and also less efficient to score when it comes to large number of flies.
In the coming posts we will discuss about use of most popular mini white gene as genetic markers for P-element vectors.
Image of rosy gene @ Dana Carroll’s lab
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