140-S08-Mar 14

140-S08-Mar 14 - How to get fly transgenes from in vitro to...

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How to get fly transgenes from in vitro to in vivo : your favorite gene(s) & w+ transposase gene A “ stable ” source of transposase, since it can’t move P-element (ends) serve as a “ vector ” to move DNA of our choice As a GENETIC SOURCE of transposase to get the transgene to insert into chromosomes use a defective (immobile) integrated P-element Inject DNA into very young embryos ….. aiming for the germ cells . modified so that it makes transposase in soma as well as germline
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whatever is useful (eg. w+ ) (a nonautonomous element) Use the engineered mobile genetic element as a mutagen (make it hop randomly into genes): 2 w+ starting element: It is a "genetically tagged" mutagen ( we can follow it )
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2 3 w+ collect progeny from the appropriate genetic cross of the dysgenic parent 2 ( not carrying the original P) Is this a mutant allele of interest? If so, already well marked and easy to clone ! 3 gene w+ starting element: new (random) site of insertion w+ transposase to mobilize (pseudo-replicative transposition) X Tp X ( not carrying the transposase source) happening In the dysgenic parent's germline
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your favorite gene(s) & w+ (a nonautonomous element) If we are going to want to use as a mutagen (hop into genes)… Lucky thing that M strains exist (strains with no pre-existing source of P transposase or antitransposase to interfere with our controlling non-autonomous element [transgene] mobility) P P
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Why do M strains exist? Lab strains taken from the wild before 1950’s don’t have P elements. This DNA parasite invaded D. melanogaster sometime in the 1950s, then rapidly spread nearest P-element relative: in a fly species 50 Myr diverged -- an example of horizontal genetic transmission (xfrd in same generation , i.e. gene not introduced from parents (vs. vertical) How? Are fly-workers to blame for contaminating D. melanogaster ?
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collect progeny from the dysgenic parent How do we know that we have even mutated a gene, much less generated a mutant allele of use/interest ?
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140-S08-Mar 14 - How to get fly transgenes from in vitro to...

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