Generating a Drosophila melanogaster mus109 Rescue Construct
Session Title
Biology and Genetics
College
College of Arts and Sciences
Department
Biology
Faculty Mentor
Kathryn Kohl, Ph.D.
Abstract
DNA is constantly being repaired in vivo, but this repair process can only occur through the action of DNA repair pathways. Previous studies in our lab have molecularly mapped the Drosophila melanogaster gene mus109. To confirm that we have located the correct gene, we need to conduct a rescue experiment. This inserts a wild-type version of the gene of intrest into the mutant genome of Drosophila melanogaster to see if it reverses the mutant phenotype. Therefore, the goal of this project was to create the necessary rescue construct. To do this we used the Gateway cloning system (Invitrogen), a method based on the lambda phage recombination system, to insert the candidate gene’s cDNA into two destination vectors. One of the destination vectors contains the gene’s stop codon, while the other does not. The former construct was designed to produce an HA-tagged C-terminal fusion protein. Both constructs were verified by restriction enzyme digest and Sanger sequencing and are currently being purified from bacteria. Upon reaching the proper concentration, the DNA for both vectors will be injected into fly embryos. If inserting a wild-type copy of the candidate gene into the mus109 mutant background allows those flies to repair damaged DNA—unlike mus109 mutants—then we will have confirmed that our candidate gene is indeed mus109.
Course Assignment
BIOL 471, 472 – Kohl
Grant Support?
Supported by an SC INBRE grant from the National Institute for General Medical Sciences (NIH-NIGMS)
Start Date
12-4-2019 3:15 PM
Generating a Drosophila melanogaster mus109 Rescue Construct
WEST 219
DNA is constantly being repaired in vivo, but this repair process can only occur through the action of DNA repair pathways. Previous studies in our lab have molecularly mapped the Drosophila melanogaster gene mus109. To confirm that we have located the correct gene, we need to conduct a rescue experiment. This inserts a wild-type version of the gene of intrest into the mutant genome of Drosophila melanogaster to see if it reverses the mutant phenotype. Therefore, the goal of this project was to create the necessary rescue construct. To do this we used the Gateway cloning system (Invitrogen), a method based on the lambda phage recombination system, to insert the candidate gene’s cDNA into two destination vectors. One of the destination vectors contains the gene’s stop codon, while the other does not. The former construct was designed to produce an HA-tagged C-terminal fusion protein. Both constructs were verified by restriction enzyme digest and Sanger sequencing and are currently being purified from bacteria. Upon reaching the proper concentration, the DNA for both vectors will be injected into fly embryos. If inserting a wild-type copy of the candidate gene into the mus109 mutant background allows those flies to repair damaged DNA—unlike mus109 mutants—then we will have confirmed that our candidate gene is indeed mus109.
