Knock-in mouse model with a non-sense mutation in the Ppt1 gene.

Not specified

To construct the targeting vector, a 5′ homology arm (∼1.9 kb) and a 3′ homology arm (∼5.2 kb) of the mouse Ppt1 gene (GenBank accession# NP_032943.2) were generated by PCR using mouse genomic DNA as the template and high fidelity Taq DNA polymerase. The primers used to generate the 5′ arm fragment were as follows: Forward: 5′- acg ttc gc GAC TGC TCT TCT GAA GGT CCT GAG TTC A-3′, Reverse: 5′- acg tgg cgc gcc ata TGA GAG AGC CAG GTC CTA TGG AGC T-3′. The primers used to generate the 3′ arm fragment were as follows: Forward: 5′-acg tcc gc GGG ACA CAG ACA AGC ATG TAG TCA AAA C-3′, Reverse: 5′- acg tgc ggc cg CAG AAT GTC CCA AAC CAT GCT C-3′. The amplified PCR fragments were subcloned into a cloning vector (FV vector, Taconic, Cranbury, NJ). The c.451C>T (R151*) mutation was introduced into the 5′ arm of exon 5 of the mouse Ppt1 gene using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). The primers used for site-directed mutagenesis were as follows: Forward: 5′-GGT GTC TTT GGA CTC CCC TGA TGC CCA GGA GAG AGT TCT-3′, Reverse: 5′-AGA ACT CTC TCC TGG GCA TCA GGG GAG TCC AAA GAC ACC-3′. The recombinant plasmids were confirmed by restriction digestion and DNA sequencing. In addition to the homology arms, the targeting vector also contained LoxP flanking the neomycin (Neo) resistance cassette and a DTA expression cassette. The complete nucleotide sequence of the final targeting vector was confirmed by DNA sequencing.The targeting vector was linearized by NotI and electroporated into C57BL/6J ES cells (Taconic, Cranbury, NJ). The transfected ES cells resistant to G418 were screened for homologous recombination by PCR and the correctly targeted ES cells were identified by Southern blot analysis. Two independent clones of recombinant ES cells were electroporated with a Cre-expression plasmid. The resulting deletion of the Neo cassette in recombinant, Cre-transfected clones was confirmed by PCR analysis. The correctly targeted ES clones, without the Neo cassette, were injected into BALB/c blastocysts and implanted into pseudopregnant mice. The male chimeras were mated with C57BL/6J females to generate offspring. The male and female offspring with WT/c.451C>T mutation were identified by PCR. Germline transmission of the mutation was confirmed by PCR-based genotyping and further verified by direct DNA sequencing. The breeding of the chimeric founders to generate WT/c.451C>T mice was performed by Taconic (Cranbury, NJ). Further breeding of the male and female progeny carrying the WT/c.451C>T mutation was carried out yielding WT, WT/c.451C>T, and c.451C>T/c.451C>T mice.

Wild-type littermates

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact mmrrc@missouri.edu. Older strains may not have this information.

Anil Mukherjee, NIH/NICHD.