Demecolcine化学辅助去核技术在牛体细胞核移植中的初步研究

本实验目的是研究demecolcine辅助去核的卵母细胞能否支持牛的核移植胚胎的发育。通过化学药物demecolcine处理牛MII期卵母细胞来辅助去除牛卵母细胞核,并用去核的卵母细胞做受体,进行核移植的研究。实验结果显示,demecolcine辅助去核后的卵母细胞质膜有明显一个或二个突起,并且突起内都含有卵母细胞染色体组,显示去核效果较好（57.89%～73.3%）。药物处理一小时为最适时间,去核率可达73.3%。对demecolcine辅助去核的卵母细胞的核移植胚胎发育情况显示囊胚率较盲吸法核移植胚胎较好（12.5%VS10.2%）,但二者差异不显著(p>0.05)。Demecolcine药物处理后的卵母细胞能够支持核移植胚胎的发育。Demecolcine辅助去核可以在牛体细胞核移植中的到应用。     

化学辅助去核法对绵羊卵母细胞去核率和重构胚发育率的影响

为提高绵羊体细胞核移植的效率,本研究采用一种新的去核方法—化学辅助去核法,对绵羊体外成熟的卵母细胞进行去核,研究了化学诱导剂秋水仙素的处理浓度、作用时间、卵母细胞的成熟时间对去核效果及重构胚发育的影响。结果表明:1)卵母细胞在0.4μg/mL的秋水仙素溶液中分别孵育0.5h和1h,胞质突起率和去核率没有显著的差异,突起率可高达85.4%,去核率达到100%;2)0.2μg/mL或0.4μg/mL秋水仙素溶液将卵母细胞处理0.5h,对去核效果没有显著影响;3)对于体外成熟18～23h的卵母细胞,随着成熟时间的延长,盲吸法的去核率降低,但没有影响秋水仙素诱导胞质突起的比率和去核率;4)两种去核方法对重构胚的发育没有产生显著影响,但成熟21～23h卵母细胞重构胚囊胚的发育率显著高于成熟18～20h卵母细胞重构胚囊胚的发育率。综上所述,本试验优化了绵羊卵母细胞化学辅助的去核程序,利用化学辅助去核法对高卵龄的绵羊卵母细胞进行去核,提高了去核率和重构胚的体外发育率。     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56.7%,发育至桑椹胚达11.7%、孵化囊胚率为6.7%,显著高于成纤维细胞组成的重构胚(p<0.05)。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至GO或G1期,抽吸法/解剖法采集卵母细胞,体外培养33或44 h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6-DMAP激活处理,体外培养6天,结果表明,卵母细胞采集方法、激活液中细胞松弛素(CB)并不影响重构胚的发育(以卵龄44h的卵母细胞为受体);而以电脉冲结合6-DMAP激活处理能提高重构胚发育能力(以卵龄33 h的卵母细胞为受体)(p<0.05)。本研究显示,以电脉冲结合6-DMAP激活卵丘细胞重构胚,能在体外发育至囊胚     

猪体细胞核移植重构胚的体外发育(英文)

以卵丘细胞为核供体细胞组成重构胚 ,卵裂率达到 5 6.7% ,发育至桑椹胚率达到1 1 .7% ,囊胚率为 6.7% ,显著高于成纤维细胞重构胚 (P <0 .0 5 )。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0 G1 期 ,抽吸法 解剖法采集卵母细胞 ,体外培养 3 3～ 44h ,将卵丘细胞放至去核卵母细胞的卵周隙中 ,重构胚以钙离子载体A2 3 81 7或电脉冲结合 6 DMAP激活处理 ,体外培养 6d。研究表明 ,卵母细胞采集方法、激活液中细胞松弛素 (CB)、激活程序并不影响重构胚的发育 (以卵龄 44h的卵母细胞为受体 ) ;而以电脉冲结合 6 DMAP激活处理能提高重构胚发育能力 (以卵龄 3 3h的卵母细胞为受体 ) (P <0 .0 5 )。本研究显示 ,以电脉冲结合 6 DMAP激活卵丘细胞重构胚 ,体外能发育至囊胚     

牛体细胞核移植显微操作环节的优化

本研究从牛卵母细胞去核方法(纺锤体观测仪法&Hoechst33342染色法)、供体细胞核引入去核卵细胞质的方法(卵细胞质注射法和电融合法)和重构胚胎电融合(3组参数)等3个环节对牛体细胞核移植的显微操作过程及相关参数进行了筛选优化。以核移植胚胎的卵裂率、囊胚发育率作为检测指标,对不同的方法所获得的克隆胚胎的卵分裂率与囊胚发育率进行比较,最后筛选获得1个优化的牛体细胞核移植操作程序,即采用Spindle view系统对牛卵母细胞进行去核操作,将供核体细胞注射到卵周隙,然后通过电融合法将供体核引入去核卵细胞质(电融合参数为1.9kV/cm,脉冲时程10μs,方波2次间隔2s)。以此核移植程序进行牛体细胞核移植实验,自获得克隆胚胎中筛选80枚优质囊胚移植到33头受体牛子宫内,最后2头母牛产下2头克隆牛犊,结果表明利用该优化的显微操作环节进行牛体细胞核移植可以获得体细胞克隆牛犊。     

猪体细胞核移植重构胚的体外发育

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚率达到11．7％,囊胚率为6．7％,显著高于成纤维细胞重构胚（P＜0．05）。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导G0/G1期,抽吸法／解剖法采集卵母细胞,体外培养33－44h,将卵丘细胞放至去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6－DMAP激活处理,体外培养6d。研究表明,卵母细胞采集方法、激活液中细胞松驰素（CB）、激活程度并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,体外能发育至囊胚。     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚达11．7％、孵化囊胚率为6．7％,显著高于成纤维细胞组成的重构胚（P＜0．05）。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0或G1期,抽吸法／解剖法采集卵母细胞,体外培养33或44h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电泳冲结合6－DMAP激活处理,体外培养6天,结果表明,卵 母细胞采集方法、激活液中细胞松弛素（CB）并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,能在体外发育至囊胚。     

不同供体细胞及其处理对猪核移植重构胚体外发育的影响

系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40~44 h的猪卵母细胞去核后, 将经血清饥饿(0.5%FBS)培养2~9天、0.1 mg/L Aphidicolin(APD)培养+0.5% FBS培养2~9天或一般培养法(10% FBS)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞, 直接注射到去核的卵母细胞质中, 或注射到卵周隙中, 再经电融合(100 V/mm, 30 [mu]s, 电脉冲1次)构建重构胚。重构胚以钙离子载体A23817 或电脉冲结合6-DMAP 激活处理, 体外培养6天。耳皮成纤维细胞和颗粒细胞经0.1 mg/L APD + 0.5% FBS培养处理后的重组胚卵裂率, 均高于血清饥饿和一般培养处理的同种供体细胞(P<0.01)。卵丘细胞、颗粒细胞经0.1 mg/L APD + 0.5% FBS处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P<0.05)。以猪颗粒细胞为核供体时, 电融合法的重构胚分裂率显著高于胞质内注入法(P<0.05), 但囊胚发育率无显著差异(P>0.05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞, 其具有正常二倍染色体的细胞比例均无显著差异(P>0.05); 以这2种细胞不同培养代数做供体进行核移植时, 各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P>0.05)。这些结果表明: (1) 猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定; (2) 0.1 mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果, 血清饥饿培养则无明显效果; (3) 猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞, 核移植后都能得到体细胞克隆的囊胚, 但前者的效果略优于后者, 且其核移植效果不受供核细胞培养代数的影响; (4) 电融合核移植胚胎的发育率高于胞质内直接注入法, 但两者的总体效率相近。     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

体细胞的组织来源及培养代数对猪核移植重构胚发育的影响

本研究系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40-44h的猪卵母细胞去核后,将经血清饥饿(0．5?s)培养2-9d、0．1mg/L Aphidicolin (APD)培养 0．5?S培养2-9d或一般培养法(10?S)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞,直接注射到去核的卵母细胞质中,或注射到卵周隙中。再经电融合(100V/mm,30μs,电脉冲1次)构建重构胚。重构胚以钙离子载体A23817或电脉冲结合6- DMAP激活处理,体外培养6天。耳皮成纤维细胞和颗粒细胞经0．1mg/L APD 0．5?S培养处理后的重组胚卵裂率,均高于血清饥饿和一般培养处理的同种供体细胞(P＜0．01)。卵丘细胞、颗粒细胞经0．1mg/L APD 0．5?S处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P＜0．05)。以猪颗粒细胞为核供体时,电融合法的重构胚分裂率显著高于胞质内注入法(P＜0．05),但囊胚发育率无显著差异(P＞0．05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞,其具有正常二倍染色体的细胞比例均无显著差异(P＞0．05);以这2种细胞不同培养代数做供体进行核移植时,各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P＞0．05)。这些结果表明：(1)猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定;(2)0．1mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果,血清饥饿培养则无明显效果;(3)猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞．核移植后都能得到体细胞克隆的囊胚,但前者的效果略优于后者,且其核移植效果不受供核细胞培养代数的影响;(4)电融合核移植胚胎的发育率高于胞质内直接注入法,但两者的总体效率相近。     

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.     

Spindle-view系统在猪卵母细胞去核中的应用

应用Spindle-view对体外成熟培养36、42、44和48h的猪体外成熟卵母细胞减数分裂纺锤体进行去核操作,并与传统去核方法(McGrath-Solter去核法,挤压去核法)相比较,结果表明:①在42~48h之间利用Spindle-view得到的猪卵纺锤体影像与极体的相对位置没有明显的变化;②Spindle-view适合用于猪体外成熟卵母细胞减数分裂纺锤体的观察及去核;去核效率与其他两种方法相比差异极显著(95.5%,42.1%,74.2%,P<0.01);③纺锤体成像是否清晰可用于猪卵母细胞的质量监控。     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

An epigenetic modifier results in improved in vitro blastocyst production after somatic cell nuclear transfer

The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation could significantly improve blastocyst yield compared to the control (46.4+/-4.6% vs 17.7+/-4.9% for treated and untreated embryos, respectively; p<0.05), whereas similar cleavage rate and total cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats

To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients.     

Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells

Nuclear transfer (NT) techniques have advanced in the last few years, and cloned animals have been produced from somatic cells in several species including pig. In this study we examined the feasibility of using granulosa-derived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% serum, 7 days), injected directly into cytoplasm of enucleated oocytes and the oocytes were electrically activated. The reconstructed embryos were cultured for 7 days and stained with Hoechst 33342 to determine the number of nuclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7+/-3.7% in NT and 97.4+/-4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5+/-7.2% in NT and 79.3+/-5.6% in controls. The blastocysts formation rates were 4.9+/-2.4% in NT and 26.8+/-3.8% in controls. To examine the effect of activation time on development of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post-injection. At 18 h post-activation the pronuclear formation rates were higher (62.5+/-7.3%) in the 3-4 h group as compared to the 0-0.5 h (22.0+/-12.5%) or 1-2h (44.5+/-6.3%) groups (P<0.05). However, the cleavage rates (9.6+/-4.6 to 10.7+/-4.2%) and the blastocysts formation rates (1.2+/-2.4 to 4.9+/-3.7%) were not different among treatments (P>0.05). The mean cell number of blastocysts was 15.7+/-5.7 in NT and 25.3+/-24.7 in controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that granulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocytes, and that the reconstructed embryos can develop to the blastocyst stage. In addition, EGFP can be used as a marker for gene expression of donor nuclei.     

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda-rabbit interspecies nuclear transfer

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p > 0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.