- Pre Microinjection: The location of the targeted mutation is confirmed by 3’ LR-PCR in the ES cells.
- Post injection for the mouse colony at F1 chimera progeny:
- Neo cassette count via qPCR (heterozygous mice should carry only one copy of the Neo cassette)
- Vector backbone specific PCR (2 reactions on different parts of the vector)
- Three assay short range PCR (wild type specific, mutant specific, LacZ cassette)
- Detection of homozygotes
- Neo count via qPCR
- Three assay short range PCR confirmation of homs (a small subset of qPCR homozygote samples are confirmed by srPCR, at which point the whole colony is considered to have passed QC)
In homozygous lethal lines, (minimum 28 offspring via qPCR from heterozygous x heterozygous pairings; homozygous is 0%) are cleared from QC.