Rigorous quality control protocols ensuring stable germline transmission, verified allele structure, and reproducible phenotypes
Complete sequence verification of all vector components including 5' and 3' homology arms (minimum 3-5kb each), selection cassettes, LoxP sites, and intervening sequences. Restriction digest mapping confirms expected fragment patterns. All vectors undergo Sanger sequencing across critical regions before ES cell targeting.
Southern blot analysis confirms correct integration at target locus with proper 5' and 3' junctions. PCR screening identifies positive clones, followed by long-range PCR across entire targeted region. Karyotype analysis ensures normal chromosome number and structure. Only clones passing all QC criteria advance to blastocyst injection.
Chimeric founders undergo germline transmission testing through breeding to wild type partners. F1 offspring genotyped by PCR and confirmed by Southern blot where applicable. Expression analysis (RT-PCR, Western blot) verifies expected mRNA and protein changes. Multi-generation breeding confirms stable germline transmission without mosaicism.
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