1) Which genome engineering mechanism they used to create transgenic ES (Embryonic stem cell)? What is the goal of this transgenesis?
Answer:
They chose to mutate Sos1 because it showed more expression in tissues and cell lines than Sos2. After they tested which Sos gene had more expression, they moved on to mutating Sos1 in murine ES cells using homologous integration of a lacZ-neor cassette. This cassette was placed in front of the Sos1 coding sequence. The goal was to integrate this cassette into the Sos1 locus to create a fused protein of the first 44 amino acids of the Sos1, β-galactosidase, and the neomycin phosphotransferase. These genes would be expressed under the Sos1 promoter. The goal of transgenesis was to take advantage of the higher chances of homologous recombination events that would occur using this construct where the selectable marker doesn’t have a promoter and translational initiator (this lets it use the Sos1 promoter to express these genes). A safe guard to the expression of these markers is that the vector also has the cell marker or β-galactosidase of the Sos1 expression. …show more content…
They also have paler yolk sacs, and malformation of the mid-brain and cleft palate. They examined most of these morphologies in figure 3. Upon these observations they then state that they need to observe the mutant phenotypes to their expected pattern of expression of the targeted gene. They do this by using their lacZ marker from the initial created cassette. The lacZ marker is in frame of the Sos1 promoter meaning that the expression of β-galactosidase in the heterozygous embryos will most likely be in direct correlation to expression of the Sos1 protein. The heterozygotes were stained with X-gal and a low level of β-galatosidase was observed in developing tissues, but the pattern was not helpful for locating where Sos1 protein was
Answer:
They chose to mutate Sos1 because it showed more expression in tissues and cell lines than Sos2. After they tested which Sos gene had more expression, they moved on to mutating Sos1 in murine ES cells using homologous integration of a lacZ-neor cassette. This cassette was placed in front of the Sos1 coding sequence. The goal was to integrate this cassette into the Sos1 locus to create a fused protein of the first 44 amino acids of the Sos1, β-galactosidase, and the neomycin phosphotransferase. These genes would be expressed under the Sos1 promoter. The goal of transgenesis was to take advantage of the higher chances of homologous recombination events that would occur using this construct where the selectable marker doesn’t have a promoter and translational initiator (this lets it use the Sos1 promoter to express these genes). A safe guard to the expression of these markers is that the vector also has the cell marker or β-galactosidase of the Sos1 expression. …show more content…
They also have paler yolk sacs, and malformation of the mid-brain and cleft palate. They examined most of these morphologies in figure 3. Upon these observations they then state that they need to observe the mutant phenotypes to their expected pattern of expression of the targeted gene. They do this by using their lacZ marker from the initial created cassette. The lacZ marker is in frame of the Sos1 promoter meaning that the expression of β-galactosidase in the heterozygous embryos will most likely be in direct correlation to expression of the Sos1 protein. The heterozygotes were stained with X-gal and a low level of β-galatosidase was observed in developing tissues, but the pattern was not helpful for locating where Sos1 protein was