FIGURE 2.

Conditional deletion of Jak1 in the female germline and consequential effects of JAK1 deficiency on STAT activation and embryonic development. (a) Breeding strategy to convert the floxed allele of Jak1 (Jak1^fl) into a null mutation (Jak1⁻). MMTV-Cre (line A) mice express Cre recombinase in developing oocytes. Thus, heterozygous Jak1^fl/wt MMTV-Cre females transmit a null allele to their offspring whether the Cre transgene is segregated out or not. (b) PCR assays using the primer sets illustrated in panel Figure 1a,c to validate the germline deletion of the Jak1 floxed allele and the presence of the recombined knockout/null allele of Jak1. An internal primer set for neo served as a positive control for the presence of the mutant alleles, and the amplification of a PCR fragment of the Jak2 gene was used to control for the integrity of the DNA samples. (c) Immunoblot analyses of the expression of JAKs 1 and 2 as well as the tyrosine phosphorylation of STATs 1, 3, and 6 in immortalized mouse embryonic fibroblasts (MEFs) of heterozygous and homozygous JAK1 knockouts (Jak1^wt/−, Jak1^−/−) as well as wildtype controls (Jak1^wt/wt). Beta-actin (ACTB) was used as a loading control. Immunoprecipitation (IP)/western blot analyses was used to determine the phosphorylation on tyrosine 705 and serine 727 on STAT3. (d) Comparison of JAK1 deficient embryos and littermate wildtype controls during the final stages of prenatal development. A subset of JAK1 knockout pups were alive at birth but showed symptoms of apnea. None of the pups were able to survive