To understand the function of the 10 amino acid insertion in loop 1 of myosin II heavy chain-B in the living organism, we are trying to generate transgenic mice through gene targeting (specific knock-out of these 30 nucleotides) using homologous recombination in mouse embryonic stem cells. Using mouse genomic DNA from the relevant area of the MHC-B gene, we replaced the 30 nucleotide exon with cDNA encoding neomycin. We also generated a construct containing cDNA encoding thymidine kinase which was located just outside the homologous sequence. The two mutated genomic fragments (one with neo and the other containing both neo and tk) were transfected into mouse embryonic stem cells by electroporation, in separate experiments. The clones were selected in the presence of 300 ug/ml of G418 and FIAU 0.2 uM. The selected clones were then screened by genomic Southern analysis and PCR. To date, we have identified two clones that appear to contain the neomycin construct in the proper location. These clones lacking the 30 nucleotide insert will be microinjected into blastocysts and implanted into pseudopregnant mice. As important ancillary experiments, we analyzed mouse brain mRNA using RT-PCR and found evidence for the presence of the 30 nucleotide inserted segment. Also, to be sure that we had not damaged the genomic DNA that we transfected into the mouse embryonic stem cells, we transfected a similar construct into NIH 3T3 cells and found that it could undergo the normal constitutive splicing. Thus, we should be able to generate mice with normal MHC-B protein, but which lacks the inserted isoform in their brain cells.
