Studies On Goat Induced Pluripotent Stem Cells

Induced pluripotent stem cells(iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors(OCT3/4, SOX2, KLF4, cMYC, LIN28and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs(giPSCs). The objectives of this study were that generate giPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human(h) transcription factors:hOCT4, hSOX2, hKLF4and hcMYC. On the basis of the previous research, we generated tgiPSCs using the same method. Using tgiPSCs as donor cells which detected the ability of producing cloned embryos in vitro by nuclear transfer technique. The main findings are as follows:1. Finding out the suitable induced system and culture system for giPSCsBased on the results of previous studies, the experiment used serum-free culture system "KNOCKOUT-DMEM+KSR" which was added human sources LIF, human sources β-FGF, and both of them. The results indicated that the undifferentiated state of giPSCs could not be maintained in the culture system adding LIF. Then the undifferentiated state of giPSCs could be maintained in the culture system adding β-FGF or both β-FGF and LIF.We tried to use DMEM/F12and FBS which were used in culturing livestock ESCs and iPSCs frequently. The result of experiment indicated that FBS is more suitable than KSR for culturing giPSCs. We selected primary clone for further expansion using the morphological criteria. Some clones would differentiate after2-4passages. Mechanical method were used to passage the good quality clone before5passages. Using enzymatic method, some clones is relatively large. After passaging with the enzymatic, the large clones should be devided into small clumps of cells using mechanical method. The large clump of cells was difficult to form a new clone.2. Somatic cell of transgenic cloned dairy goats induced into giPSCsOur laboratory had obtained a large batch of transgenic cloned dairy goats. The transgenic human lactoferrin(hLF) have already been integrated into the genome of them. Based on the achievement of the previous researchs, ear fibroblast cells of transgenic cloned dairy goats induced into transgenic giPSCs(tgiPSCs) harboring hLF using lentivirus. The result of experiment indicated that tgiPSCs expressed multiple pluripotent genes: OCT4, SOX2, cMYC, KLF4and NANOG; AKP reaction positive; formed embryoid body containing three mesodermal tissues in vitro; formed teratoma containing three germ layers.3. TiPSCs as donor cell is more suitable to the development of nuclear transfer embryosWe use transgenic goat skin fibroblasts(tgFs) and tgiPSCs as donor cells to produce cloned embryos(tgF-Embryos and tgiPSC-Embryos), and also collected goat embryos producing in vivo. All of the three embryos are in the8-16cell stage. We compared the expression profile of growth-promoting genes(IGF-1and IGF-2) and their receptor genes(IGF-1R and IGF-2R) in tgF-Embryos, tgiPSC-Embryos, and vivo-Embryos by using RT-PCR. IGF-1regulates the growth-promoting activity of growth hormone through IGF-1receptors. The bioactivities of IGF-2are similar to those of IGF-1, and the expression of IGF-1and IGF-2significantly affect the reprogramming of the derived embryos and their subsequent development. At the8-16-cell stage, the expression of IGF-1, IGF-1R and IGF-2R was not significantly different among tgF-Embryos, tgiPSC-Embryos and vivo-Embryos, but the expression of IGF-2was significantly lower in tgiPSC-Embryos than in tgF-Embryos. However, there was no significant difference in the expression of IGF-2in tgiPSC-Embryos and vivo-Embryos. IGF-2expression in embryos generated using NT was relatively higher than that in vivo-Embryos. The expression profile of insulin-like growth factor(IGFs) family genes(IGF-1, IGF-2, IGF-1R and IGF-2R) in tgiPSC-Embryos is closer to vivo-Embryos than tgF-Embryos.