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1.
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.  相似文献   

2.
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.  相似文献   

3.
The objective of the study was to describe and improve a technique for laparoscopic embryo transfer into the oviduct and uterine horns of pigs and to evaluate the feasibility and safety of this method. Fourteen female pigs were randomly allocated into groups A and B: three portals were used for group A, and the simulation of embryo (0.9% NaCl, 0.2 mL) was injected into the tip of uterine horn; three or four portals were used for group B, and the injection set of the oviduct was inserted through the abdominal orifice of uterine tube into the oviduct to inject the simulation of embryo. The repeat laparoscopy was performed on the 21st day. Three pigs randomly selected from each groups were repeated the same procedure three times, and then were euthanized on the 21st day after the last surgery and a complete necropsy performed. Laparoscopic embryo transfer was performed successfully in all pigs without major intra-and post-operative complications. The average surgical time which accomplished procedures for groups A and B was 18.6 min (range, 28-14 min) and 37.4 min (range, 53-29 min). Postoperatively, none of pigs appeared to abnormal signs. This study demonstrated that laparoscopic embryo transfer could be easily accomplished by using the special-purpose equipment and increasing reuse time of a female recipient.  相似文献   

4.
In order to improve reproduvtive efficieny and understand reproduvtive defense mechanism, the oviduct, uterine horn and uterine body of bovine were used to detect the changes of inflammatory factors and the relationship between estrogen and progesterone receptor protein during estrous cycle by real-time PCR and Elisa method. The results showed that interleukin-4(IL-4), interleukin-6(IL-6), interleukin-10(IL-10), interleukin-1α(IL-1α) and interleukin-1β(IL-1β) were expressed in cow oviduct, uterine horn and uterine body. In the follicular phase and the luteal phase, m RNA expression of five inflammatory factors in the uterine horn and uterine body was higher than that in the oviduct. In the follicular phase, IL-10 was highly expressed in the uterine horn and uterine body, IL-4 was highly expressed in the uterine horn, uterine body and oviduct. Additionally, in the luteal phase, IL-6 and IL-1β were highly expressed in the uterine horn, uterine body and oviduct, and the highest expression of IL-1β was observed in the uterine horn. The levels of Estrogen Receptor(ERα) protein in the oviduct, uterine horn and uterine body significantly increased in the follicular phase. The levels of Progesterone Receptor(PR) protein in the same portions of the reproductive tract in the luteal phase were significantly higher than those in the follicular phase. IL-4 and IL-10 in the cow reproductive tract might play a major role in the follicular phase, while IL-6 and IL-1β might play a major role in the luteal phase. The expression of five inflammatory factors was not directly regulated by ERα and PR.  相似文献   

5.
In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium (mCZB) as control group. The experimental group (Ica group) was supplemented with 0.6μmL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21: pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group ((85.14?.57)% vs. (72.04?1.58)%; (82.50?.11)% vs. (66.80?1.70)%, respectively, P<0.01). The total cell number of blastocysts had extreme difference between Ica group and control group ((61.40?.64) vs. (46.23?.50), P<0.01). The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group ((1.47?.51) vs. (2.94?.66); (2.40?.27)% vs. (6.25?.62)%, respectively, P<0.01). Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 (P<0.01), down-regulated pro-apoptotic Caspase3 (P<0.05) and PTEN (P<0.01), up-regulated anti-apoptotic Bcl-2 (P<0.01). In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro-apoptotic genes. These apoptosis-related genes were regulated by miRNA-21.  相似文献   

6.
Factors affecting the efficiency of nuclear transfer （NT） in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses （P〈 0.05）, but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups （P〉0.05）. When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos （P〈 0.05）, although the fusion rate did not significantly differ among the three groups （P〉 0.05）. Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage （P 〈 0.05）, although the fusion rate was similar （P 〉 0.05）. Activation of cytoplasts prior to fusion increased the cleavage rate （P〈 0.05） and blastocyst development （P〈 0.05） of reconstructed embryos, but decreased the fusion rate （P 〈 0.05） compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM ＋ 3% OCS at the first 48 h and then cultured in TCM199＋ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199＋ 10% FCS or TCM199＋3% OCS （P 〈 0.05）. When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.  相似文献   

7.
[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturation and embryo development in vitro were always added into 1% ITS(Insulin-transferrin-selenium) or using 1% ITS to replace FBS in 2 kinds culture solutions for conducting in vitro cultures of goat oocyte and parthenogenetic embryo.The influences of ITS on their developments were detected.[Result] ITS in maturation liquid of oocytes could not increase oocytes maturation rate but significantly increased blastocyst rate (58.06% vs. 48.19%)of parthenogenetic embryo.If FBS in maturation liquid of oocytes was replaced by ITS, the maturation rate, cleavage rate and blastocyst rate were basically unchanged.Adding ITS into embryo medium could increase blastocyst rate (68.30% vs. 56.82%)of parthenogenetic embryo of goat.If FBS in embryo medium was replaced by ITS,the cleavage rate didn’t change basically,while the blastocyst rate in ITS was obviously lower than that in FBS group(42.33% vs.56.82%).[Conclusion] ITS could promote maturation of oocyte in vitro and early embryonic development, in addition,ITS could replace serum in maturation medium of oocyte as serum-free culture system for conducting relevant researches.  相似文献   

8.
This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.  相似文献   

9.
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.  相似文献   

10.
[ Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [ Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [ Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells （ FF： 53.33% vs. 10.00%; MGE： 33.33% vs. 6.67%）. Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture （ FF： 53.33% vs. 10.00% ; MGE ： 33.33% vs. 6.67% ）, confluence time of amplification culture was significantly decreased （20 -30 d）. The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [ Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.  相似文献   

11.
[目的]探讨供体细胞类型、移植胚胎发育阶段、数量及部位对山羊转基因克隆效率的影响。[方法]利用体细胞核移植技术将转染人乳铁蛋白基因hLF的山羊胎儿成纤维细胞(GFF)和乳腺上皮细胞(GMGE)移植到MII期去核卵母细胞内,经电融合、激活、体外培养后,2~8细胞期克隆胚被移植到同期发情山羊的输卵管内,囊胚被移植到子宫角内。[结果]GFF与GMGE的妊娠率相近(输卵管移植妊娠率分别为26.47%及20.00%);在GFF,输卵管移植的妊娠率与子宫角内移植妊娠率接近(分别为26.47%及25.00%),输卵管移植胚胎平均数为21.2组的妊娠率显著高于5.93组和9.64组(40.00%及26.67%,21.43%)。[结论]供体细胞类型、移植胚胎的发育阶段及移植部位对山羊转基因克隆效率的影响不大,但对于输卵管移植,受体羊移植胚胎数量对妊娠率有明显的影响。此外,该研究还提示了利用成年羊乳腺上皮细胞制作转基因动物的可行性。  相似文献   

12.
采胚方法对波尔山羊胚胎移植效果的影响   总被引：8，自引：1，他引：7  
采用2种采胚方法研究了64只波尔山羊的胚胎移植效果。结果表明:输卵管采胚法的胚胎回收率极显著高于(P<0.01)子宫角采胚法,且前者的获胚数与可用胚数均显著高于(P<0.05)后者,而两者的黄体数则差异不显著(P>0.05),说明采用输卵管采胚法所采集的胚胎数量与质量均优于子宫角采胚法;而子宫角移胚法的受体妊娠率及产羔率均极显著高于(P<0.01)输卵管移胚法。试验还表明,输卵管采胚法对处理供体术后的发情率有一定的不良影响,故在山羊胚胎移植中应优先采用子宫角采胚法回收胚胎。  相似文献   

13.
【目的】探讨分析兔胚胎移植的影响因素,为提高兔胚胎移植效率及生产转基因兔奠定基础。【方法】采集获得的交配胚胎经体外培养后,按单只受体移植胚胎数、受体类型（经产母兔与青年母兔）、受体同期发情方式随机分组,对比观察胚胎移植后受体母兔的妊娠情况。【结果】单只受体兔每次移植10～20枚和20～30枚胚胎获得的妊娠率分别为23．1％和20．0％,二者差异不显著（P〉0．05）;采用经产母兔为受体可获得显著高于青年母兔的妊娠率（27．8％vs13．3％,P〈0．05）;以自然同期发情为受体母兔胚胎移植后获得的妊娠率显著高于诱导同期发情的受体母兔（30．7％vs10．0％,P〈0．05）。【结论】兔胚胎移植时选择自然发情的经产母兔作为受体能获得较好的胚胎移植效果,单只受体移植胚胎数以控制在10-30枚为宜。  相似文献   

14.
胚胎移植方法和受体母猪因素对克隆猪生产效率的影响   总被引：2，自引：0，他引：2  
【目的】建立高效的胚胎移植技术体系,以提高克隆猪的生产效率。【方法】比较不同移植方法和不同受体母猪状况的胚胎移植分娩率和克隆猪的出生效率,利用体外发育能力相似的不同品种和发育阶段的克隆胚胎混合移植确定最佳的受体母猪发情同期时间。【结果】克隆胚胎经由输卵管伞移植比输卵管打孔移植具有更高的移植分娩率和克隆猪效率（2.2% 和0.4%,P＜0.01）,而胚胎移植后辅助人工授精会降低克隆效率（0.6% 和 2.2%,P＜0.01）。利用经产母猪作为移植受体比青年猪受体具有更高的克隆效率（3.0%和0.8%,P＜0.01）和平均窝产仔数（（5.5±0.7）头和（2.7±0.3）头,P＜0.05）。受体母猪发情时间比胚胎激活时间晚12-36 h组的克隆效率分别为显著（P＜0.05）和极显著（P＜0.01）,高于晚0 h和早12-24 h 组的克隆效率（2.0%、0.5%和0%）。最佳的受体母猪发情时间为晚于克隆胚胎激活后24 h,克隆效率达3.0%。【结论】选择自然发情时间晚于克隆胚激活后24 h的经产母猪为受体,通过输卵管伞端移植胚胎,能够获得较高的克隆效率,是猪克隆胚胎移植的较理想方法。  相似文献   

15.
【目的】利用体细胞核移植技术克隆陕北白绒山羊优秀种公羊个体。【方法】以特别优秀成年陕北白绒山羊种公羊耳部皮肤成纤维细胞为供体细胞,体外培养成熟的陕北白绒山羊卵母细胞作为受体,利用显微操作方法对成熟卵母细胞进行去核操作,然后将供体细胞注射到其卵周隙内,经电融合将其导入去核卵母细胞内形成核移植重组胚。利用钙离子载体Ionomycine联合蛋白酶抑制剂6甲基氨基嘌呤（6-DMAP）对核移植重组胚进行激活处理,挑选激活后完整的胚胎继续进行体外培养,然后在2～16细胞期时将克隆胚胎移植到相应的同期发情受体母羊体内,利用PCRRFLP技术鉴定克隆羊。【结果】构建了体细胞核移植胚胎253枚,重组胚胎的融合率为71.54%(181/253),体外培养后的卵裂率为68.33%（123/180）,囊胚发育率为25.20%（31/123）;在此基础上,构建了537枚克隆胚,将其中卵裂的359枚分别移植到32只代孕母羊体内,移植60 d后,有2只受体母羊确认妊娠,最终1只受体母羊妊娠第4月流产出2只死羔;另1只受体母羊维持到期并顺产体细胞克隆羊1只。经遗传学鉴定,2只流产羊羔与1只存活个体均为体细胞核移植后代。【结论】获得陕北白绒山羊克隆个体,建立了相对完善的陕北白绒山羊克隆技术体系。  相似文献   

16.
曾文滔  张爱华  吕萌  刘燕 《安徽农业科学》2013,(27):11053-11055
[目的]提高ES细胞囊胚注射的移植出生率。[方法]对照组采用正常囊胚ES细胞注射,试验组注射后的囊胚经酸性台氏’液消化透明带后直接移植,研究Es细胞囊胚注射后脱透明带移植对小鼠产仔率和嵌合率的影响。[结果]对照组产仔率为15．96％,嵌合率为57．89％。试验组小鼠产仔率为17．95％,嵌合率为57．14％,出生率提高2％。[结论]嵌合率无显著差异,酸性台氏液消化透明带的方法可简化操作流程。  相似文献   

17.
采用FSH(促卵泡素 )结合LH(促黄体素 ) ,以 2种组合分别对 1 9只和 1 4只两组供体山羊进行超排处理 ,得到 541枚的排卵结果 ,共回收胚胎 41 1枚 ,受精卵数为 32 0枚。 2种处理方法分别平均取得了 1 8.4枚和 1 3 .6枚的排卵结果 (p <0 .0 5)。将 32 0枚受精卵中的 1 92枚2 ,4和 8细胞胚胎通过手术法分别移植到 96只受体山羊输卵管内 ,每只受体移植 2枚胚胎 ,3种胚龄的胚胎移植后的妊娠率分别为 68.4% ,74.1 %和 84.0 % (p >0 .0 5)。  相似文献   

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