蚜肠霉素处理及细胞传代数对小型猪体细胞核移植胚胎发育的影响

doi:10.3969/j.issn.1674-5817.2011.05.007

摘要/Abstract

摘要： 目的为优化小型猪体细胞核移植技术(SCNT)技术,探讨供体细胞不同类型、不同传代数以及蚜肠霉素(Aphidicolin,APC)处理各供体细胞对重构胚体外发育的影响。方法小型猪输卵管上皮细胞(OEC),耳成纤维细胞(EFC)和卵丘细胞(CC)分别经血清饥饿或血饥饿后再使用0.1 ?g/ml APC处理。核移植重构胚体外培养7 d, 以检测其发育能力。结果 OEC经APC处理后, 重构胚的卵裂率和囊胚形成均显著高于仅血清饥饿处理组(61.82% vs 56.25%, 24.55% vs 17.86%; P<0.05)。以EFC为供体细胞,APC组重构胚卵裂率显著高于血清饥饿组(63.36% vs 57.01%; P<0.05)。注入CC,APC处理能显著提高重构胚的囊胚形成率(29.63%; P<0.05)。三种细胞经APC处理,CC作为核供体,重构胚的卵裂率和囊胚形成率最高。三种细胞中,无论哪一种作为核供体,其传代数对重构胚的发育没有影响。结论以CC为供体细胞,血清饥饿后再使用APC处理,更适合小型猪的体细胞核移植。

关键词: Aphidicolin, 小型猪, 细胞类型, 传代数, 体细胞核移植

Abstract: Objective To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types, cell passage number and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Methods The Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 ?g/ml APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Results Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs 56.25%, 24.55% vs 17.86%; P<0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs 57.01%; P<0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocysts formation rate by APC treatment (29.63%; P<0.05). In the presence of APC, CC gave rise to highest cleavage and blastocyst formation rates among the three cell types. All of the three donors can be used as donor karyoplasts for nuclear transfer, and the efficiency is not influenced by culture passages. Conclusion The treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

Key words: aphidicolin, minipig, cell type, passage number, somatic cell nuclear transfer.

中图分类号:

Q95-33

张廷宇, 戴建军, 张树山, 吴彩凤, 张德福. 蚜肠霉素处理及细胞传代数对小型猪体细胞核移植胚胎发育的影响[J]. 实验动物与比较医学, 2011, 31(5): 338-344.

ZHANG Ting-Yu, DAI Jian-Jun, ZHANG Shu-Shan, WU Cai-Feng, ZHANG De-Fu. Effects of Different Donor Cells Treatment with Aphidicolin and Donor Cell Passages on the Preimplantation Development of Somatic Cell Nuclear Transfer Embryos in Minipig[J]. Laboratory Animal and Comparative Medicine, 2011, 31(5): 338-344.

参考文献

[1] Wilmut I, Schnieke AE, Mcwhir J, et al.Viable offspring derived from fetal and adult mammalian cells[J]. Nature, 1997, 385(6619):810-813.
[2] Kato Y, Tani T, Sotomaru Y, et al.Eight calves cloned from somatic cells of a single adult[J]. Science, 1998, 282(5396): 2095-2098.
[3] Wakayama T, Perry AC, Zuccotti M, et al.Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J]. Nature,1998, 394(6691):369-374.
[4] Baguisi A, Behboodi E, Melican DT, et al.Production of goats by somatic cell nuclear transfer[J]. Nat Biotechnol,1999, 17(5):456-461.
[5] Polejaeva IA, Chen SH, Vaught TD, et al.Cloned pigs produced by nuclear transfer from adult somatic cells[J]. Nature, 2000, 407(6800):86-90.
[6] ChesnéP, Adenot PG, Viglietta C, et al. Cloned rabbits produced by nuclear transfer from adult somatic cells[J]. Nat Biotechnol, 2002, 20(4):366-369.
[7] Shi D, Lu F, Wei Y, et al.Buffalos (Bubalus bubalis) cloned by nuclear transfer of somatic cells[J]. Biol Reprod, 2007, 77(2):285-291.
[8] Zhang YH, Pan DK, Sun XZ,et al.In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells[J]. Zygote, 2006, 14(3):239-247.
[9] Kraqh PM, Nielsen AL, Li J, et al.Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer’s disease-causing dominant APPsw[J]. Transgenic Res, 2009, 18(4):545-558.
[10] Hoshino Y, Uchida M, Shimatsu Y, et al.Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig[J]. Cloning Stem Cells,2005, 7(1):17-26.
[11] Hao YH, Yong HY, Murphy CN, et al.Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets[J]. Transgenic Res, 2006, 15(6):739-750.
[12] Miyoshi K, Inoue S, Himaki T, et al.Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment[J]. Mol Reprod Dev, 2006, 74(12):1568-1574.
[13] Zhao J, Ross JW, Hao Y, et al.Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer[J]. Biol Reprod, 2009, 81(3):525-530.
[14] Li Y, Liu J, Dai J, et al.Production of cloned miniature pigs by enucleation using the spindle view system[J]. Reprod Dom Anim,2010, 45(4):608-613.
[15] Wang TS.Eukaryotic DNA polymerases[J]. Annu Rev Biochem 1991, 60:513-552.
[16] Batchelder CA, Hoffert KA, Bertolini M, et al.Effect of the nuclear-donor cell lineage, type, and cell donor on development of somatic cell nuclear transfer embryos in cattle[J]. Cloning Stem Cells, 2005, 7(4):238-254.
[17] 张德福, 刘东, 汤琳琳, 等. 卵母细胞去核方法的改进[J]. 中国兽医学报, 2006, 26(5):574-577.
[18] 张廷宇, 戴建军, 吴彩凤, 等. 钙离子及山梨醇对猪卵母细胞孤雌激活的影响[J].中国兽医学报, 2010, 30(3):406-410.
[19] Egli D, Birkhoff G, Eggan K.Mediators of reprogramming: transcription factors and transitions through mitosis[J]. Nat Rev Mol Cell Biol, 2008, 9(7):505-516.
[20] Wakayama T, Yanagimachi R.Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei[J]. Reproduction, 2001, 122(1): 49-60.
[21] Heyman Y, Zhou Q, Lebourhis D, et al.Novel approaches and hurdles to somatic cloning in cattle[J]. Cloning Stem Cells, 2002, 4(1):47-55.
[22] Oback B, Wells D.Donor cells for nuclear cloning: many are called, but few are chosen[J]. Cloning Stem Cells, 2002, 4(2): 147-168.
[24] Mitalipov SM, Zhou Q, Byrne JA, et al.Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling[J]. Hum Reprod, 2007, 22(8): 2232-2242.
[25] Tani T, Kato Y, Tsunoda Y.Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming[J]. Biol Reprod, 2001, 64(1): 324-330.
[26] Collas P, Pinto-Correia C, Ponce de Leon FA, et al. Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos[J]. Biol Reprod, 1992, 46(3):501-511.
[27] Kues WA, Anger M, Carnwath JW, et al.Cell cycle synchronization of porcine fetal fibroblasts: Effects of serum deprivation and Reversible cell cycle inhibitors[J]. Biol Reprod, 2000, 62(2): 412-419.
[28] Goissis MD, Caetano HV, Marques MG, et al.Effects of serum deprivation and cycloheximide on cell cycle of low and high passage porcine fetal fibroblasts[J]. Reprod Domest Anim, 2007, 42(6): 660-663.
[29] Kues WA, Carnwath JW, Paul D, et al.Cell cycle synchronization of porcine fetal fibroblasts by serum deprivation initiates a nonconventional form of apoptosis[J]. Cloning Stem Cells, 2002, 4(3):231-243.
[30] Kurosaka S, Nagao Y, Minami N, et al.Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts[J]. Biol Reprod ,2002, 67(2):643-647.
[31] Peterson CL, Logie C.Recruitment of chromatin remodeling machines[J]. J Cell Biochem, 2000, 78(2):179-185.
[32] Roh S, Shim H, Hwang W, et al.In vitro development of green flourescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts[J]. Reprod Fertil Dev, 2000, 12(1-2):1-6.
[33] Cho J, Bhuiyan MM, Shin S, et al.Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell[J]. J Vet Med Sci, 2004, 66(12): 1567-1573.
[34] Kubota C, Yamakuchi H, Todoroki J, et al.Six cloned calves produced from adult fibroblast cells after long-term culture[J]. Proc Natl Acad Sic USA, 2000, 97(3):990-995.
[35] Wells DN, Misica PM, Tervit HR.Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells[J]. Biol Reprod, 1999, 60(4): 996-1005.
[36] Capecchi MR.How close are we to implementing gene targeting in animals other than the mouse?[J]. Proc Natl Acad Sci USA, 2000, 97(3): 956-957.
[37] Cho SR, Ock SA, et al.Effects of confluent, roscovitine treatment and serum starvation on the cell-cycle synchronization of bovine foetal fibroblasts[J]. Repord Dom Anim, 2005, 40(2): 171-176.
[38] Kurome M, Ishikawa T, Tomii R, et al.Production of transgenic and non-transgenic clones in miniature pigs by somatic cell nuclear transfer[J]. J Reprod Dev, 2008, 54(3): 156-163.
[39] 马育芳, 卫恒习, 李燕, 等. 化学激活和季节对克隆猪出生率的影响[J].中国科学C辑: 生命科学, 2009, 39(4):384-391.
[40] Lee JW, Wu SH, Tian XC, et al.Production of cloned pigs by whole-cell intracytoplasmic microinjection[J]. Biol Reprod, 2003, 69(3):995-1001.
[41] Park KW, Lai LX, Cheong HT, et al.Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts[J]. Biol Reprod, 2002, 66(4):1001-1005.
[42] Yin XJ, Tani T, Yonemura I, et al.Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes[J].Biol Reprod, 2002, 67(2): 442-446.
[43] Tomii R, Kurome M, Ochiai T, et al.Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes[J]. Cloning Stem Cells, 2005, 7(4):279-288.
[44] Oropeza M, Petersen B, Carnwath JW, et al.Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli[J]. Xenotransplantation, 2009, 16(6):522-534.
[45] Koo OJ, Hossein MS, Hong SG, et al.Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer[J]. Zygote, 2009, 17(1):37-43.