共查询到20条相似文献，搜索用时 984 毫秒
1.
以水牛原生殖细胞（PGCs）为核供体,成熟水牛卵母细胞为受体,采用电融合法和胞质内直接注核法对水牛PGCs的核移植进行了研究。从水牛胎儿的生殖嵴或生殖腺分离得到PGCs,在胎儿成纤维细胞饲养层上传代培养后,进行核移植。结果显示,当采用电融合法时,PGCs核移植的融合率、分裂率、囊胚率和总囊胚率均显著高于胎儿成纤维细胞（P〈0．05）;当采用直接注核法进行核移植时,PGCs的核移植囊胚率显著高于胎儿成纤维细胞（P〈0．05）。但分裂率差异不显著（P〉0．05）。结果表明,水牛PGCs细胞是理想的核移植供体细胞。 相似文献
2.
本研究利用自制的兔抗牛脾细胞免疫血清裂解供体细胞膜而获得核胞体,结果表明,自制的免疫血清可以成功的裂解供体细胞膜而获得核胞体。将该法制备的核胞体应用于牛体细胞核移植的胞质内注射中,从而为核移植中供体细胞核胞体的获得提供一种新的思路。在比较不同供体细胞对重构胚发育的影响时。结果表明。颗粒细胞与耳成纤维细胞构建的重构胚卵裂率没有显著差异（62．62％versus67．89％,P〉0．05）,囊胚率也没有明显的差异（23．88％ versus 18．92％,P〉0．05）：在4℃低温冷藏一定时间的供体细胞与未经冷藏的供体细胞,其卵裂率和囊胚率都没有显著性差异（63．34％versus62．03％;26．67％versus24．45％,P〉0．05）;同样,耳成纤维细胞也得到相同的结果（70．76％verSus66．67％：21．74％versus19．56％,P〉0．05）。颗粒细胞构建的重构胚在注核后3～5h进行激活较1～3h有更高的卵裂率．表现出显著性差异（73．91％versus56．52％,P〈0．05）,裳胚率无显著性差异（23．53％,versus15．38％,P〉0．05）。 相似文献
3.
供体细胞对猪体细胞克隆胚胎早期发育的影响 总被引：1，自引：1，他引：1
以中国农业大学实验用小型猪香猪胎儿成纤维细胞、成年耳成纤维细胞和颗粒细胞3种细胞系为供体细胞进行核移植。比较了血清饥饿法和接触抑制法处理胎儿成纤维细胞诱导进入G0／G1期的效率，发现二者差异不显著（P〉0．05），血清饥饿2d和4d差异不明显，同样接触抑制2d和4d差异也不显著（P〉0．05）。系统研究了影响克隆胚胎发育的供体因素：血清饥饿与否、细胞形态、细胞类型及个体差异等，结果表明：血清饥饿处理对克隆胚的早期发育没有明显的促进作用；圆形光滑细胞有利于细胞融合，对早期发育无显著影响（P〉0．05）；不同个体、不同类型的供体细胞对克隆胚囊胚发育率有一定的影响。 相似文献
4.
优化山羊体细胞核移植方案的研究 总被引：4，自引：1，他引：4
以乳腺细胞、胎儿成纤维细胞和颗粒细胞为供体细胞进行核移植，比较了供体细胞类型、细胞冷冻及核移植的重建方法（胞质内注射、电融合）对重构胚早期发育的影响。结果：（1）乳腺细胞支持重组胚的发育能力（囊胚率7．14％）显著低于成纤维细胞（16．19％）和颗粒细胞（19．01％）（P〈0．05），卵裂率无显著差异（P〉0．05）；（2）乳腺上皮细胞构建重组胚时电融合法的效率（69．52％）极显著高于胞质注射法（59．20％）（P〈0．01）；成纤维细胞的重组成功率、卵裂率和囊胚率在两种方法间均无显著差异（P〉0．05）；在颗粒细胞，胞质注射法重组成功率（82．17％）显著高于电融合法（50．99％）（P〈0.05），其囊胚率也稍高于电融合法，但无显著差异（P〉0.05）；（3）上述3种供体细胞在冻融后支持重组胚发育力方面无显著差异（P〉0.05）。结论：在山羊体细胞核移植中，上述3种细胞支持重组胚的发育力以成纤维细胞和颗粒细胞较好；细胞冷冻对其无显著影响；在构建重组胚方法上，乳腺细胞以电融合法为宜，颗粒细胞是胞质注射法优于电融合法，而成纤维细胞两种方法均可。 相似文献
5.
首先获得绵羊胎儿成纤维细胞、成年耳组织成纤维细胞及前脂肪细胞,用脂质体介导法、BTX电转法及Nucleofector核转法对上述细胞系转染绿色荧光蛋白(gfp)基因,并对转染效率进行比较,最后探讨3种转gfp基因细胞对绵羊转基克隆胚发育的影响。结果:核转法的转基因效率最高,3种细胞的转基因效率分别为86%,87%和90%,显著高于BTX电转法和脂质体法基因转染效率;脂质体转染效率显著低于BTX电转效率(P<0.05)。而对同一种转染方法而言,基因转染效率在3种细胞间差异均不显著(P>0.05)。在以上述3种转基因细胞为核供体的转基因克隆试验中,胚胎融合率、卵裂率均无显著性差异(P>0.05),胎儿成纤维细胞组的囊胚率显著高于前脂肪细胞组(P<0.05),但耳组织成纤维细胞组与胎儿成纤维细胞、前脂肪细胞间差异均不显著(P>0.05)。结果表明核转法基因转染效率最高,胎儿成纤维细胞最适合于胚胎克隆研究。 相似文献
6.
7.
体细胞核移植生产转ω-3脂肪酸去饱和酶基因(sFat-1)的猪胚胎 总被引：1，自引：1，他引：0
本研究通过脂质体介导的方法将来源于线虫C.Briggsae的ω-3脂肪酸去饱和酶基因（sFat-1）转染至大白猪胎儿成纤维细胞。采用600μg·mL^-1G418药物浓度,经连续10 d筛选及PCR、RT-PCR鉴定,获得11个转基因阳性细胞克隆。以体外成熟42 h的猪卵母细胞与转基因细胞构建重构胚。经体外培养后观察,转基因克隆胚胎与非转基因胚胎的卵裂率（76.6%±4.1%vs.81.6%±3.1%）和囊胚率（10%±1.97%vs.9.7%±1.4%）均无显著差异（P〉0.05）。采用放线菌酮进行化学二次激活时,胚胎的囊胚率显著高于采用电二次激活胚胎（20.6%±0.89%vs.10%±1.97%,P〈0.05）,但二者的卵裂率并无显著差异（72.4%±4.96%vs.76.6%±4.1%,P〉0.05）。研究表明,通过脂质体介导的方法,可以获得转sFat-1基因大白猪胎儿成纤维细胞系;以该细胞系为核供体构建的转基因克隆胚胎与非转基因克隆胚胎的发育能力无显著差异;二次激活采用放线菌酮进行化学激活能够显著提高胚胎的囊胚发育率。 相似文献
8.
本试验通过比较供体成纤维细胞的不同血清饥饿培养天数、传代次数、冻存复苏等方面试验条件对重组胚发育的影响因素进行了深入的研究。结果表明：供体细胞血清饥饿0、1～3、4～6、7～9d之间重组胚卵裂率没有显著差异（P〉0.05），但囊胚率以饥饿1～3d的最高，与4～6和7～9d组别差异显著（P〈0．05）；以传代0、1～3、4～6、7～9代的细胞作为供体细胞，卵裂率没有显著差异（P〉0.05），桑椹胚率以传1～3代最高，差异显著（P〈0．05）；2代细胞解冻后的卵裂率显著高于4和8代细胞，而以2和6代冷冻解冻后细胞为供体，卵裂率并无明显差异，8代细胞的桑椹胚率显著低于其它组。本试验为提高供体成纤维细胞在卵母细胞中重新程序化及后续重组胚发育等相关研究提供参考。 相似文献
9.
猪冷冻精液体外受精研究 总被引：2，自引：1，他引：1
为简化猪体外受精过程中精液的来源,避免不同公猪精液和不同采精时间对猪体外受精的影响,本研究采用5mL大管冷冻精液进行体外受精（IVF）,研究IVF过程中的各项参数。结果表明：精卵共孵2～8h对多精人卵率影响不大（P〉0．05）,2-细胞率随着共孵时间的延长而增加,8h时达到72．8％,显著高于2h组（60．5％,P〈0．05）,桑椹胚和囊胚发育率以6h组最高,分别达46．1％和3．48％。多精人卵率随精子浓度的增加而增加,且各组之间差异显著（P〈0．05）,2-细胞率也以10^8组（81．2％）最高,且显著高于10^5组（63．7％）（P〈0．05）,同时10^7组和10^8组分别获得了8．11％和3．13％的囊胚发育率。以NCSU-23作为成熟培养液,并部分去除卵丘细胞的IVF卵裂率和囊胚率最高,达77．1％和7．33％。冻精和新鲜精液的IVF,无论是在卵裂率,还是桑椹胚或囊胚率上均无显著差异（P〉0．05）。 相似文献
10.
小鼠皮肤成纤维细胞的体细胞核移植 总被引：1，自引：1，他引：0
取成年小鼠唇部皮肤进行培养，分离成纤维细胞并血清饥饿培养1周，用作核供体。对成年小鼠进行超排，取卵母细胞用作核受体，核移植重构胚经SrCl2激活处理6h后，同mM16培养液和小鼠输卵管上皮细胞共培养，把发育到早期囊胚的重构胚转移至小鼠胎儿成纤维细胞饲养层上，添加ES细胞条件培养液，消化分离ICM，然后接种培养，对孵出的ES细胞样集落进行鉴定培养。结果显示，小鼠唇部皮肤成纤维细胞为核供体，核移植重构胚2-细胞率为54．05％，桑椹胚率17．14％，囊胚率6．90％，对照组卵丘细胞的核移植重构胚2-细胞率为60．00％，桑椹胚率21．85％，囊胚率11．69％，但2种供体细胞在支持核移植重构胚发育能力上差异不显著。成纤维细胞重构囊胚中6个囊胚分离出ES细胞样集落，3个ES细胞样集落可稳定传代；对照组卵丘细胞重构囊胚中9个囊胚中分离出ES细胞样集落，5个ES细胞样集落可稳定传代。从核移植重构胚中分离出的ES细胞样集落具有岛状或巢状群体生长形态，生长旺盛的集落可自发分化成单个散在或片状存在的上皮样或梭形细胞，碱性磷酸酶检测为阳性，常规冻存复苏，仍显示ES细胞特征。 相似文献
11.
The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development. 相似文献
12.
Joohyeong Lee Yongjin Lee Geun‐Shik Lee Seung Tae Lee Eunsong Lee 《Reproduction in domestic animals》2019,54(9):1258-1264
Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC‐derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC‐derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF‐derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell‐oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC‐derived SCNT embryos showed higher blastocyst formation (48.4%) than FF‐derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT. 相似文献
13.
影响猪ICSI转基因技术效率的主要因素研究 总被引：2，自引：0，他引：2
以猪体外成熟卵子和冷冻解冻的死精子为材料,以pEGFP-N1为模式基因,探讨注射台温度、激活后6-DMAP的处理和精子与PEGFP-N1孵育液添加BSA(牛血清白蛋白)对精子胞质内注射(ICSI)转基因效率的影响。结果表明:注射台温度为30℃时的阳性率为40.07%,而38.5℃时为20.97%,差异极显著(P<0.01)。添加BSA的囊胚转基因率为55.56%,对照组为33.33%,差异极显著(P<0.01)。6-DMAP处理组与对照组的转基因率分别为52.53%和26.25%,差异极显著(P<0.01);而且6-DMAP处理组的囊胚率(9.96%)显著高于(P<0.05)对照组(2.30%)。研究表明注射台温度对转基因效率有明显影响,温度高转基因率低;精子与PEGFP-N1孵育液添加BSA对转基因胚胎发育有一定促进和保护作用,有利于提高囊胚转基因率;激活后用6-DMAP处理能提高转基因率和囊胚率。 相似文献
14.
The influence of human growth hormone (hGH) on the differentiation of preadipocytes was examined in primary cultures of stromal-vascular (s-v) cells from porcine adipose tissue. In these experiments, cells were exposed to test media for 7–8 days after seeding and plating for two days in fetal bovine serum. In serum-free (insulin, transferrin and selenium) cultures hGH (1 and 10 nM) reduced the number and size of fat cell clusters (P<.05) by 50% relative to controls (no hGH). Differentiation of preadipocytes was assayed by labelling dividing cells with tritiated thymidine under identical conditions and then exposing cultures to test media for seven days. Fat cells were then separated from the other cells and radioactivity was determined in each fraction. In serum containing (2% pig serum) cultures hGH (10 nM) inhibited (P<.05) the differentiation of labelled preadipocytes. In cultures with serum and with 1 μM insulin and in serum-free cultures, 1 and 10 nM hGH reduced (P<.05) the levels of glycerol phosphate dehydrogenase (GPDH) specific activity by approximately 50%. However, hGH (1 and 10 nM) had no affect on GPDH activity in cultures with serum but without insulin. These studies indicate that hGH significantly impedes porcine preadipocyte development in vitro. Therefore, the decreased rate of adipose tissue growth observed in pigs chronically treated with GH could be due in part to impaired preadipocyte growth. 相似文献
15.
The relationships between adipocyte and muscle cell development within muscle are important in the study of factors or agents that may improve meat quality. Neonatal porcine muscle has the potential to yield both cell types for cell culture because it contains developing adipocytes and a high number of muscle satellite cells. Therefore, we modified a conventional collagenase-based procedure to digest neonatal porcine muscle and subsequently cultured the resultant muscle stromal-vascular (SV) cells on several substrata in basal and dexamethasone (DEX)-containing media. Developing myotubes and preadipocytes were present in muscle SV cell cultures on laminin substrata following seeding and plating with fetal bovine serum (FBS) with or without DEX. Myotube number was much higher (P < 0.05) on laminin substrata compared with all other substrata, whereas preadipocyte number in muscle SV cell cultures was independent of substrata, as we have shown previously. This approach can be used to establish co-cultures of differentiating adipocytes and myotubes from collagenase-digested neonatal pig muscle. Because the comparison is within the same culture dish, this method allows for a direct comparison of the responses of adipogenic and myogenic cells to growth and differentiation factors. For example, DEX did not alter myogenesis (i.e., 11 +/- 3 vs. 11 +/- 4 myotubes per unit area for control and DEX-treated cultures, respectively), but it has been shown to markedly increase preadipocyte number in muscle SV cell cultures. 相似文献
16.
试验利用水牛卵泡液(BuFF)和黄牛卵泡液(BoFF)对不同来源水牛卵母细胞体外受精效果的影响进行了探讨,以完善水牛体外受精培养系统,进一步提高水牛胚胎体外生产效率。试验按成熟培养液中添加卵泡液替代胎牛血清量共分4个组。不添加卵泡液(0%+10%胎牛血清)为Ⅰ组(对照组);添加5%卵泡液+5%胎牛血清为Ⅱ组;添加10%卵泡液+0%胎牛血清为Ⅲ组;添加15%卵泡液+0%胎牛血清为Ⅳ组。结果表明,添加BuFF对活体采集卵母细胞和屠宰场收集卵母细胞的体外受精卵分裂率无显著影响(P0.05),但添加5%和10%BuFF对卵母细胞体外受精后的胚胎发育有明显促进作用,囊胚率均极显著高于对照组和15%BuFF组(P0.01),5%和10%BuFF组间无显著差异(P0.05);添加15%BuFF囊胚率有降低的趋势,但与对照组相比差异不显著(P0.05)。而添加10%BoFF组活体采集卵母细胞体外受精的受精卵分裂率和囊胚率均极显著高于对照组和5%BoFF组(P0.01),添加5%BoFF组的受精卵分裂率和囊胚率与对照组无显著差异(P0.05);添加BoFF对屠宰场收集水牛卵母细胞体外受精卵分裂率无显著差异(P0.05),但添加5%和10%BoFF组的囊胚率均显著高于对照组和15%组(P0.05),添加15%BoFF组与对照组相比,囊胚率显著降低(P0.05),5%和10%BoFF组间囊胚率无显著差异(P0.05)。综合以上结果,在水牛卵母细胞成熟培养液中添加5%～10%的BuFF或BoFF代替牛血清,可明显提高水牛体外胚胎生产效率,且以添加同种的BuFF效果略好。 相似文献
17.
以CZB为基础培养液,培养小鼠4、8-细胞胚胎单卵裂球,研究葡萄糖、牛磺酸和猪输卵管上皮细胞共培养在其体外发育中的作用。结果表明:牛磺酸添加与否对4、8-细胞胚胎单卵裂球的囊胚发育率分别为29%、30%和14%、14%,无显著性差异(P>0.05)。添加葡萄糖后,4、8-细胞胚胎单卵裂球的囊胚发育率分别为36%和19%,有显著提高(P<0.05)。含有葡萄糖而牛磺酸的添加与否对4、8-细胞胚胎单卵裂球的囊胚发育率分别为36%、38%和19%、20%,无显著性差异(P>0.05)。各组内4、8-细胞胚胎单卵裂球形成的囊胚细胞数分别为(10.44±1.24～(12.43±1.18)和(7.57±0.97)～(8.48±1.16),均无显著性差异(P>0.05)。4、8-细胞胚胎单卵裂球与猪输卵管上皮细胞共培养,其囊胚发育率分别为47%和26%,囊胚细胞数分别为(17.57±1.13)和(11.43±0.92),均高于单一培养(P<0.05)。 相似文献
18.
水牛体细胞核移植方法比较及激活前的时间间隔对全细胞胞质内注射法核移植效果的影响 总被引：1，自引：0，他引：1
本研究旨在比较水牛2种体细胞核移植(Somatic Cell Nuclear Transfer,SCNT)方法的效果以及激活前的时间间隔对全细胞胞质内注射法(Whole-Cell Intracytoplasmic Microinjection,WCICSI)核移植效果的影响.采用水牛胎儿成纤维细胞作为供核,比较了透明带下注核法(Perivitelline Microinjection,PM)和WCICSI核移植效果.另外,试验了不同类型的供体细胞进行全细胞胞质内注射后与激活前的受体胞质的最适宜作用时间.结果,WCICSI构建核移植重构胚的成功率显著高于PM(87.1%vs 81.1%,P＜0.05),虽然其重构胚的分裂率极显著低于PM(49.5%vs 71.8%,P＜0.01),但囊胚率、核移植的效率无显著差异(P＞0.05).卵丘细胞和胎儿成纤维细胞在注射后3 h激活,重构胚的囊胚发育率最高;颗粒细胞注射后与激活前的最佳时间间隔可在1.5～3 h,但3 h是最佳的作用时间.结果表明,(1)WCICSI可用于水牛体细胞核移植的研究;(2)水牛胞质内注射供体细胞后3 h进行激活,核移植重构胚的发育效果最好. 相似文献
19.
为了提高猪孤雌囊胚贴壁率,试验从饲养层及培养液两方面研究猪孤雌囊胚贴壁能力;用小鼠、猪和牛的胎儿成纤维细胞制作饲养层,分别添加DMEM、NCSU-23、DMEM/NCSU-23培养液,探讨猪孤雌囊胚在3种饲养层上的发育效果。结果表明,BEF饲养层能更好地促进猪孤雌囊胚贴壁生长,其囊胚贴壁率为33.67%,与MEF饲养层组的囊胚贴壁率(19.08%)之间差异显著(P0.05),与PEF饲养层组之间囊胚贴壁率差异不显著(P0.05),MEF饲养层组和PEF饲养层组之间囊胚贴壁率差异不显著(P0.05);在BEF牛胎儿成纤维细胞饲养层组,用猪胚胎培养液NCSU-23培养猪孤雌囊胚后,囊胚贴壁率(22.53%)显著高于DMEM培养液组(10.41%)和DMEM/NCSU-23培养液半量混合组(12.05%)(P0.05),DMEM培养液组和DMEM/NCSU-23培养液半量混合组之间差异不显著(P0.05)。牛胎儿成纤维细胞饲养层和猪胚胎培养液NCSU-23能更好地促进猪孤雌囊胚后期贴壁。 相似文献
20.
《Research in veterinary science》2013,94(3):1190-1194
Intramuscular fat (IMF) content plays an important role in meat quality. Triglyceride (TG) metabolism in intramuscular adipocytes is strongly associated with the intramuscular fat deposition. To better understand the mechanisms leading to IMF deposition we compared the expression levels of genes related to preadipocyte differentiation and lipogenesis in the intramuscular preadipocytes isolated from the longissimus muscle of Wujin and Landrace pigs. The results showed that the intramuscular preadipocytes could differentiate into mature adipocytes in vitro. Triglyceride content in adipocytes isolated from Wujin pigs was higher than Landrace pigs during the middle and later phases of preadipocyte differentiation. The expression levels of genes related to preadipocyte differentiation such as PPARG and CEBPA showed differential expression between Wujin and Landrace porcine adipocytes during the early stage of differentiation. The expression levels of lipogenic genes such as FASN and SREBF1 were significantly higher in Wujin porcine intramuscular preadipocytes than in Landrace intramuscular preadipocytes at the middle and the later stages of differentiation. This suggests that preadipocyte differentiation and lipogenesis exhibited breed-related scheduling. 相似文献