Molecular Biology Of Porcine Leukemia Inhibitory Factor

Leukemia inhibitor factor (LIF) is a multifunctional cytokine belonging to interleukin-6(IL-6) family. It was first found as a myeloid leukemic cell proliferation suppressor anddifferentiation inducer cytokine. The natural form of LIF is a 38 to 67 kDa glycosylatedprotein widely expressed in both adult and embryonic eutherian mammals . Numerous in vitroand in vivo studies indicated that LIF exhibits various important biological functions ininducing myeloid leukemic cell differentiation, maintaining embryonic stem (ES) cellpluripotency, and in hippocampal development, blastocyte implantation, etc. One of the mostfascinating role is its ability of inhibiting mouse embryonic stem (ES) cell differentiation invitro, thus maintaining the mouse ES cells in a pluripotent status. This gave rise to a newtechnology in life science-gene targeting, which have made it possible to modify or disrupt aselected single gene in ES cells and get germ line-transmitted mutant mouse colonies byembryonic manipulation. The gene-modified mouse models revolutionized thecharacterization of gene functions in vivo. In the past 20 years thousands of gene knockoutand knockin mouse models have been successfully generated and used as powerful tools tostudy gene function. The challenge is that there is so far no successful example in germ-linetransmission in model animals other than mouse despite that significant efforts have been puton developing germline-transmmitable stable pluripotent ES cells from larger animals such asrat, rabbit, pig, sheep, and cattle.Studies across eutherian mammals showed that LIF and its gene share high levels ofsimilarity among species. Many endeavors have been done to maintain ES or embryonic germ(EG) cells of other species with mouse LIF or mouse embryonic feeder cells, but none wassuccessful. A recent study has shown that mouse LIF alone is not sufficient to maintainchicken EG in a pluripotent or even undifferentiated status . Other cytokines such as chickenstem cell factor, bovine basic fibroblast growth factor, human IL-11 and insulin-like growthfactor-1 may be needed as well.The organ physiology of pig is very similar to human in many aspects, thus making it anideal model for studying human physiology and disease mechanisms. Stable pluripotentporcine ES cells are not successfully established so far. Many reasons might account for thefailures. The reality that none of the studies used porcine LIF in the culture of porcine ES orEG cells makes it reasonable to presume that porcine LIF would be much more effective inmaintaining porcine ES or EG cells in the pluripotent status than its mouse counterpart.However, molecular cloning of the gene encoding porcine LIF has not been documented.The purpose of this study is to clone the cDNA sequence encoding porcine LIF (pLIF),to express recombinant pLIF protein in prokaryotic and eukaryotic expression system and tocharacterize the activity of recombinant pLIF proteins and immunolocalization of pLIF . A606 bp full-length pLIF cDNA encodes a 202 amino acid protein was cloned by RT-PCR.pLIF has 84% and 86% amino acid sequence identity to mouse and human LIFs, respectively.The deduced amino acid sequence of pLIF protein contains six conserved consensus N-linkedglycosylation sites and six cysteines to form potential disulfide bonds. The recombinant pLIFwas expressed in E coli. as its mature form (unglycosylated protein in inclusion body) and inCHO cells as a secreted form (glycosylated natural form in the conditioned medium). Bothforms of the recombinant pLIFs can maintain murine embryonic stem cells in anundifferentiated state in culture. The recombinant pLIFs will be used to explore long-termculture of germline-transmmitable pluripotent porcine embryonic stem cells suitable forgene-targeting. In addition, immunostaining has shown that pLIF was expressed in spleniccord and monolayer covering epithelium and was expressed in myocardial cell.