1. Mate animals (donor) containing genetic region of interest (transgene, knock-out gene, etc.) to animals of recipient strain to produce the F1 generation. Recipient animals must be inbred and homozygous at the locus of interest. All F1 animals will be 100% heterozygous at every locus.
2. Mate F1 animals to animals of recipient strain. We do not use genetic markers on the X or Y chromosomes in our assay therefore it is recommended that the sex chromosomes be fixed early in the breeding scheme for convenience. Mate female F1 animals to male recipient strain animals to fix the Y and generate the N2 generation.
3. Analyze the DNA from 8-10 males who carry the genetic region of interest by microsatellite analysis. We offer genotyping services for performing or developing gene-specific PCR assays to genotype for the genetic region of interest or the client can perform the genotyping prior to sending tail snips for microsatellite analysis.
4. Based on the microsatellite analysis, you choose 2-4 animals that have the greatest percentage of recipient genome and mate these to female recipient strain animals. This will generate the N3 generation and fix the X chromosome in the males.
5. Analyze the DNA from 8-10 males with the genetic region of interest from the N3 generation by microsatellite analysis. Choose the 2-4 males that have the greatest percentage of recipient genome. Continue this selected breeding backcross process through the N5 generation. At this point, >99% of the genome should be derived from the recipient genetic background.