Identification of Hemostatic Modifier Genes Rescuing FVII Deficient Mice

FVII^tta/− mice are heterozygous for the FVII- null allele and a gene-targeted allele expressing very low levels of FVII. These mice produce very low levels of FVII (<1.0 % of wt levels). FVII^+/− and FVII^tta/+ mice have each independently been backcrossed into a C57Bl/6 background (thus all the mice in the lineage have had at least 50% of wt FVII levels and there is no selection for compensation for low coagulant activity). The FVII⁻ and FVII^tta alleles have similarly been backcrossed extensively into a 129X1/svj background. The FVII^tta/− mice were generated in both C57Bl/6 and 129X1/svj backgrounds by crossing FVII^+/− and FVII^tta/+ animals. In the C57Bl/6 background these low FVII mice suffer intrauterine or perinatal lethality. In contrast FVII^tta/− mice generated in a 129X1/svj background survive much longer with 50% surviving more than 6 months. These results indicate there are significant strain differences in the phenotype of mice with a FVII_tta/− genotype. To determine the genetic basis of these strain differences we have generated F1 FVII^tta/− mice in a 50:50 C57Bl/6 and 129X1/svj background by crossing C57Bl/6J FVII^+/tta and 129X1/svj FVII^+/− parents. These mice survive long-term similar to FVII^tta/− in a pure 129X1/svj background. F1 FVII^tta/− males were backcrossed with C57Bl/6J FVII^+/− females to generate F1B1 FVII_tta/− males. Long-term surviving (>16 weeks) FVII^tta/− males were selectively backcrossed with C57Bl/6J FVII^+/− females to generate F1B2, F1B3 and then F1B4 FVII^tta/− mice. Presumably, the survival of these FVII^tta/− mice depends upon the retention of 129X1/svj derived chromosomal regions enabling the survival of low FVII mice. After 4 rounds of backcrossing, the F1B4 FVII^tta/− mice retain ~3% of the 129X1/svj genome. DNA from 6 F1B3 FVII^tta/− males and all their 81 F1B4 FVII^tta/− offspring (total of 87 mice) have been genotyped with a Mouse MD Linkage panel (Illumina) containing 247 informative SNPs distinguishing between 129X1/svj and C57Bl/6J genomes. The initial scan identified 12 chromosomal regions enriched for 129X1/svj derived sequences. To identify putative hemostatic modifier genes, long-term surviving F1B4 low FVII mice have been selectively backcrossed and the DNA of their offspring analyzed with a high density SNP scan in the regions of interest.