Important Genes In Human Fetal Liver Source Pact Gene Targeting Studies

Liver is the major gland in human body. It functions in metabolism of many substances. The fetal liver during 22 weeks of gestation can be considered as a source of hematopoietic stem cells. It is a major site of embryonic hematopoiesis and immune development in human. Therefore, the fetal liver may contain lots of novel genes with important functions. We have performed a large-scale sequencing of cDNAs isolated from a fetal-liver (22 weeks of gestation) cDNA library and established the expression profile of human fetal liver aged 22 weeks of gestation. Many novel genes with important functions are being studied, including PACT, HPO, CKIP-1, NPDC-1, LMBP and so on.Gene targeting is a kind of technique by which target gene can be modified accurately in the designed manner. In this study, we constructed 3 targeting vectors. Among them, HPO, PACT and CKIP-1 targeting vectors were electroporated into mouse embryonic stem cells (ESC) and the targeted clones were identified by PCR and Southern blotting. Targeted ES cells were injected into blastocysts and 3 lines of viable chimeras were yielded. HPO and CKIP-1 chimeras did not transfer the mutant allele to their offspings. PACT chimeras produced viable and fertile heterozygotes without overt phenotype. Heterozygous mice were intercrossed, and the offsprings were genotyped by PCR analysis. No viable PACT^/- mice were identified.Embryos from heterozygous intercrosses were analyzed at different days of gestation. We found the homozygotes died around embryonic days 6.5-7.5. Mutant embryos were found developmentally retarded and failed to undergo gastrulation. These results demonstrated the important role of PACT in mouse early embryonic development. By using semi-quantitative RT-PCR for E7.5 embryos, we found transcription of p21 and Mdm2 was dramatically increased. PACT interacts with p53 in vivo and inhibits its transcriptional activity in vitro. It is possible that inactivation of endogenous PACT could enhance the expression of p53 target genes, then lead to growth retardation and embryonic lethality. Further analysis of PACT-deficient mice is under investigation.