Name: ______________________________ ID: _______________________________1 1. a. kan tet kan - cut wolf DNA and plasmid DNA with EcoRI, - mix together and ligate the wolf DNA and plasmid DNA fragments, - transform the ligated DNA into bacteria (that are kansand tets) - grow on kan to select for bacteria that have a plasmid - of these bacteria, those that cannot grow on tet are the bacteria containing plasmids with an insert of wolf DNA b. Identify the wolf DNA clone(s) that are homologous to the dog gene by colony hybridization using the dog gene as a probe. 2. a. The first step is to produce the targeting vector. The neomycin gene is inserted into a protein encoding/exon region of gene D, thereby making it unable to produce functional protein. The vector construct also has the thymidine kinase (tk) gene. Next this targeting vector is introduced into embryonic stems cells. When homologous recombination occurs between the targeting vector and the genomic D gene in the embryonic stem cells, the region of the targeting vector that has gene D with the neomycin gene insertion replaces the endogenous gene. When insertion is random/non-homologous/ectopic instead of homologous, all of the vector, ie tk and D with neomycin, is inserted into random locations in the embryonic stem cell genome. Finally the embryonic stem cells are screened for homologous insertions. In order to tell which have the targeted mutation, the embryonic stem cells are grown on both neomycin and ganciclovir. Neomycin kills all of the cells that do not have the targeting vector inserted somewhere in the genome. Ganciclovir kills all of the cells that have the tk gene inserted. Thus, the embryonic stem cells that have a targeted mutation in gene D can grow on neomycin and ganciclovir. The embryonic stem cells that have a random insertion grow on neomycin, but are killed by ganciclovir. Embryonic stem cells with no insertion cannot grow on neomycin . b. (4 pts) Briefly describe how you would use the embryonic stem cells derived in part a to produce a transgenic mouse carrying the gene D mutation. Embryonic stem cells that have a targeted/knockout mutation in gene D are injected into a developing mouse embryo that has genes for a different coat color than those carried by the embryonic stem cells. (e.g agouti/brown coat color for the embryonic stem cells, and black coat color for the host embryo). This embryo then continues to develop into a mouse, and the coat color indicates whether or not the mouse consists partly of embryonic stem cells.
has intentionally blurred sections.
Sign up to view the full version.