Clone And Expression Of Mouse Leukemia Inhibitory Factor And Research On Maintaining Chicken Embryonic Stem Cells In The Undifferentiated State

ES (Embryonic Stem) cells were first isolated in cultures of inner cell mass of preimplantation embryos or Primordial Germ cells. In long vitro culture, ES cells can be maintained an undifferentiated state and ES cells have very important effects on the development biology, the clinic medicine, the animal breeding, the molecular genetics and so on, which causes the alluring foreground of application.EGFP gene was cloned and constructed secretive eukaryotic expression vector by digestion, ligation and transformation to study the transfection efficiency of pSecTag in chicken embryonic fibroblast cells. The expression of the EGFP gene was observed under fluorescence microscope after 24h or 48h, and the content of EGFP was meared in medium. It was efficient to transfect embryonic fibroblast cells of chicken with the method of lipofectamine mediation. Analysis with SAS software, we concluded that these was a obvious positive correlation between the quantity of EGFP expression and the quantity of expression vector added in medium(P<0.01), but there was no positive correlation between the quantity of EGFP expression and the quantity of lipofectamine added in medium. The optimum condition of transfection was with 2ul lipofectamine and lug expression vector in medium. The experiment established the foundation for the target gene expression in embryonic fibroblast cells of chicken.Mouse secreted LIF cDNA with and without signal peptide were cloned from mouse livers by RT-PCR, and then subcloned into pBS-T vector and pMD18-T simple vector. Digesting fragments were recovered and inserted into pSecTag/HygroB. Restrictive enzymes digestion analysis and DNA sequence results revealed that the LIF gene was cloned into eukaryotic expression vector pSecTag/Hygro successfully. The expression vectors constructed were called pSecTag-mlif(sp~+) and pSecTag-mlif(sp~-). The sequence encoding mouse LIF gene cloned is consistent with published sequence in GenBank except that the encoding sequence had two mutations with T replaced G in 216bp and in318bp. Using DNAMAN software, we found that the two mutations did not affect protein translation. Transfect CEF with pSecTag-/M///fr/?+,) and pSecTag-mlif(sp~) by the method of lipofectamine mediation, collected supernate after centrifugating cellular medium. We surveyed the mLIF expression in cellular medium with the experiment of ELISA and western-blot, but the level of expression for mLIF with pSecTag-mlif(sp~) was obvious higher than with pSecTag-mlif(sp+).As the table 2-2 showing, we cultured CES cells with nine different methods including three control groups, which all utilized CEF as feeder. The CES clones cultured in the forth group and the sixth group proliferated perfectly and the seventh is the next. The second passage CES clones cultured in the third group and the fifth group proliferated very well at first, but the CES clones began differentiating three days later. The number of CES cells clone cultured in the first group and the second group was not many, and there were many dead cells in the second passage. We found that the number of CES cells clones was more than the control groups(P<0.01), and the number of CES cells clones in different groups was significantly different.The cDNA encoding chicken LIF was cloned by the method of mRNA subtraction and RACE recently. The putative chLIF protein includes the six cysteine residues conserved in mammalian LIFs. The number of asparagineS'linked glycosylation sites was the same, but the position of three of them differed between chLIF and the mammalian LIFs. The chicken LIF precursor protein has 42.7% and 39.3% indentity with human and mouse LIF, respectively. Thus homologue between chicken and mammalian LIFs is low as with other interleukins (Hiroyuki et al, 2004) . Designing primers according to the sequence (BD187371) in NCBI, we cloned cDNA encoding chLIF. We found a novel 122bp sequence, which was inserted into coding sequence of chLIF. There was no homologous DNA sequence with the 122bp sequence by blast in NCBI, and there was no homologous amino acid sequence. In the light of the bioinformatic analysis we conferred that the novel122bp sequence should be caused by alternative splicing, and we should study the mechanism of the alternative splicing further. We also found that using the same pair of primers, two fragments were amplified in white Leghorn, but a fragment was amplified in Shouguang black chicken. We did not know the relation ship between chLIF and chicken production performance, but the result of the experiment established the foundation and provided a new idea for further study.