Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes.
Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.
Objective To determine whether arsenic has estrogen-like effects,the cell proliferation was measured iil human eervical cancer line(HeLa)in vitro.Methods The HeLa cells were grown in improved RPMI 1640 supplemented respectively with β-estradiol(E2,1 nmol/L),Arsenic trioxide(As2O3,0.5,1.0,5.0 μmol/L),ICI (500 nmol/L),E2(1 nmol/L)+ICI(500 nmol/L),As2O3(1.0 μmol/L)+ICI(500 nmol/L)and control.The growth morphology of HeLa cell was observed under microscope after 72 h.The method of M1Tr was used to study the cell proliferation after 24.48 and 72 h.The technique of flow eytometry was used to measure cell cycle after 48 h. Results HeLa cells in E2 and 0.5 μmoL/L As2O3 treatment were more better growth in morphology than control group.Percentage of HeLa cells proliferation at 24,48,72 h in E2 and 0.5 μmol/L As2O3 treatment were 6.35%, 11.56%,38.33%and 6.35%,8.50%,20.26%respectively.The proliferation effect of HeLa cells was similar in two treatments.The proliferation of HeLa cells were inhibited in other treatments.Compared with control[(41.68± 1.05)%],HeLa cells were promoted go to S phases in E2[(55.72±2.31)%]and 0.5 μmol/L As2O3[(47.82± 1.41)%]treatment.But in other treatments HeLa cells were hold back to S phases.Compared with control,there was a significant differenee(P＜0.05)of cell percentage in S phases in 5.0 μmol/L As2O3[(21.11±4.99)%]and ICI[(20.16±4.76)%]treatments.Conclusion Small amounts of As2O3 impose estrogen.1ike effects and stimulate the proliferation of HeLa cells.
Key words:
Arsenic; Estrogens; HeLa cells
Application of drugs made in China on culture of embryonic stem cells
could decrease the cost of ES cell research. In our experiment,
mytomycin made in China（Zhejiang Hisun Pharmaceutical Co.,Ltd,
authorized H33020786）was used to process feeder cells, from
which mouse embryonic stem cells have been isolated and cultured in
vitro. When the blastocysts were cultured directly on treated feeder
cell layer, the inner cell masses were growing. After 4-6 days culture,
ICM-derived outgrowths were separated into clumps, and placed on
mitomycin C-treated mouse embryonic fibroblasts feeder layer. Every 2-3
days, passage were made ever since. These ES cells have been proven to
have a potential for germ-line contribution.
【Objective】The present study aims at constructing a eukaryotic expression vector pCDsRed2-KV of goat VEGF164(vascular endothelial cell growth factor) gene and then to transfer it into Inner Mongolia Cashmere Goat(Capra hircus) fetal fibroblast(GFb) cells to obtain a transgenic cell clones,which stably expresses red fluorescence and expresses VEGF164 in hair follicle cells specifically.The transgenic cell clones can be used for nuclear transplantation.【Method】pCDsRed2-KV(6.3 kb),a hair-follicle-specific expression vector of VEGF164,was constructed by connecting VEGF164 gene to downstream of KAP6-1 promoter,and then inserting the KAP6-1 promoter-VEGF164 gene fragment into the basic vector pCDsRed2,which contains a DsRed expression unit.The Inner Mongolia Cashmere Goat fetal fibroblast(GFb) cells were transfected with the expression vector by lipofectamineTM2000.Transgenic cell clones were obtained after screening by G418.The recombinant of exogenous DNA was identified by polymerase chain reaction.【Result】The sequencing result showed that the VEGF164 gene was connected properly to the downstream of pKAP6-1,then the CMV promoter and the DsRed2 gene in sequence.Exogenous DNA in the cell clones was examined by PCR and the promoter KAP6-1 as well as VEGF164 gene has been integrated into GFb cells genome stably.【Conclusion】A hair-follicle-cell-specific expression vector of VEGF164 gene was constructed successfully and transfered into GFb cells.These data provide a way to obtain the transgenic goat by nuclear transfer in the future.
To study the expression of connexin43(Cx43) and connexin45(Cx45) in sheep preimplantation embryos,RT-PCR was used to detect the mRNA level of the 2 connexins, and their cDNA were sequenced.Ovine immatured oocytes,matured oocytes and embryos from 2-cell stage to blastocyst produced in vitro were recovered,and RT-PCR was used to semi-quantify mRNA expression.Proteins of Cx43 and Cx45 were detected by using a combination of immunocytochemistry and Laser Scanning Confocal Microscope.Cx43 and Cx45 were expressed in oocytes and preimplantation embryos at mRNA and protein le-(vels.) The 2 connexins distributed mainly on the surface of cell membrane and could not be detected in the nuclei and cytoplasm.The results provided foundation for future studies of functions of Cx43 and Cx45 in sheep preimplantation embryos.
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