不同血清对水牛早期胚胎性别比率的影响

研究了不同血清对水牛早期胚胎性别比率的影响.结果表明,同种血清不同批次对胚胎性别比率没有影响.添加胎牛血清组3个不同发育阶段雄性胚胎比率之间差异显著(P<0.05),随着细胞数目的增加雄性胚胎比率不断增加.添加发情牛血清组3个不同发育阶段雄性胚胎比率之间差异显著(P<0.05),但不同于胎牛血清组,未出现随着细胞数目的增加而雄性比率不断增加的现象.无血清组两个细胞期雄性胚胎比率之间差异不显著(P>0.05).     

不同激活方法对水牛ICSI介导转基因效果的影响

本研究探讨了不同激活方法对水牛ICSI介导转基因效果的影响。水牛体外成熟卵进行ICSI介导转基因(ICSI-mediated gene transfer,ICSI-Tr)操作后,先用5μmol/L的离子霉素(ionomycin,Ion)激活处理5min,然后分成3组,再分别用10μg/mL放线菌酮(cycloheximide,CHX)培养5h(CHX5h组)、10μg/mLCHX培养3h后再用2mmol/L的二甲氨基嘌呤(6-DMAP)培养2h(CHX3h-DMAP2h组)或用培养液(CM)培养3h后再用2mmol/L6-DMAP培养2h(CM3h-DMAP2h组)。结果显示,CHX5h组的分裂率、早期胚胎基因表达率和囊胚发育率最高,且显著高于CM3h-DMAP2h组(66.7%比49.5%,p<0.05;66.7%比40.0%,p<0.01;22.2%比9.9%,p<0.05)。ICSI18h后检查原核形成率,发现CHX5h和CHX3h-DMAP2h处理组的完整精子头百分率显著低于CM3h-DMAP2h处理组(p<0.05),2原核(pronucleus,PN)百分率则显著提高(42.4%比23.8%,p<0.05;44.6%比23.8%,p<0.01)。以上结果表明,在水牛ICSI介导转基因时,Ion联合CHX较Ion联合6-DMAP激活重构胚效果好。     

胚胎冷冻技术在转基因动物中的应用

胚胎冷冻技术在转基因动物中的应用李劲松,成国祥,李厚达（扬州大学农学院生物技术研究所,江苏省生物工程重点实验室）１９８０年,Ｇｏｒｄｏｎ首次将外源基因导入小鼠受精卵而获得成活个体,从而开了转基因动物的先河。两年后,Ｐａｌｍｉｔｅｒ等将重组的大鼠生长激...     

以精子为载体的体外转基因猪胚胎研究

去精清精子与外源DNA共孵育,使其携带外源DNA,再与成熟卵母细胞进行体外受精,生产转基因猪胚胎,为通过胚胎移植生产转基因猪奠定基础。结果:通过PCR从不同时期胚胎中检测出携带外源DNA的阳性胚,说明精子与外源DNA共孵育能使精子携带外源DNA,并通过体外受精技术使外源DNA进入早期胚胎。     

精子介导法生产转基因牦牛杂种胚胎的研究

研究外源DNA转染对牦牛精子体外受精(IVF)、早期胚胎发育以及绿色荧光蛋白(GFP)基因在早期胚胎表达的影响。用构建融合人乳铁蛋白基因绿色荧光蛋白表达载体(pAcGFP1-hLF)转染的牦牛精子IVF黄牛卵母细胞,生产的胚胎在荧光显微镜下观察GFP表达情况。结果显示,用环形质粒、线性质粒pAcGFP1-hLF转染的精子及未转染精子IVF后的卵裂率分别为56.4%、54.8%和61.0%,线性转染的囊胚率显著低于环形转染(17.7%与35.7%)。环形质粒转染的精子用DNaseⅠ消化、不消化及未转染的精子IVF后卵裂率分别为44.8%、55.2%和56.3%,囊胚率分别为33.3%、33.3%和30.2%。未经DNaseⅠ消化洗涤的环形质粒组GFP胚胎阳性率显著高于线性质粒组(99.3%与76.6%);DNaseⅠ消化组GFP胚胎阳性率极显著低于不消化组(25.4%与99.3%)。结果表明,用环形质粒DNA转染牦牛精子不会影响其受精及早期胚胎发育能力,但DNaseⅠ消化洗涤显著降低GFP胚胎阳性率。     

转基因技术对粮食生产的影响

介绍了转基因技术对粮食生产的诸多影响,尤其强调了转基因技术在解决日益膨胀的人口吃饭问题上的重要作用。应该坦然面对转基因技术,在津津乐道转基因技术给我们带来的种种好处的同时,也必须对其安全性给予足够的关注。     

我国培育世界首例冷冻胚胎克隆水牛

近日,广西水牛研究所利用克隆胚胎冷冻技术,使一头杂交母水牛顺利产下一头冷冻胚胎移植的体细胞克隆雌性水牛犊。冷冻胚胎克隆水牛犊在世界尚属首例,这是继1月1日广西水牛研究所利用国外优质种牛和本地水牛成功克隆出世界首例亚种问水牛之后的又一重大研究成果。该项目为农业部“948”项目。     

转基因克隆动物技术及其应用

克隆动物是目前生物技术领域研究的热点之一,其科学意义和应用价值重大。根据现有理论和技术发展趋势,本文提出了转基因克隆动物的概念,即将转基因动物技术与克隆动物技术有机地结合,认为转基因克隆动物制作技术有望成为２１世纪创建遗传工程动物的主导性技术。     

冷冻对山羊精子转染外源DNA和体外制备转基因胚胎的影响

本实验将鲜精和冻精分别与地高锌标记的线形化的pEGFP-N,质粒孵育转染,用原位杂交方法检测转染效率;PCR和Southern Blotting检测精子与外源DNA的整合效率;与成熟卵母细胞体外受精,PCR检测阳性胚胎比率,用透射电镜技术、碘化丙锭和羟化荧光素双探针技术和单细胞电泳（Single Cell Gel Electrophoresis,SCGE）技术,观察精子冷冻前后的超微结构、精子质膜完整性和精子核DNA损伤的变化,研究冷冻对山羊（Caprahircus）精子转染内化外源DNA和体外制备转基因胚胎的影响及机理。结果表明,冻精显著提高了转染外源DNA的效率（81．60％&#177;16．59％VS32．95％&#177;2．93％,t=4．873,P=0．003;41．80％&#177;6．26％vs27．89％&#177;8．64％,t=2．634,P=0．039）。PCR和Southern Blotting检测表明外源DNA已经整合到精子基因组上。用冻精与成熟卵母细胞体外受精,体外受精穿透率和卵裂率显著低于鲜精组（24．19％&#177;3．15％vs58．86％&#177;3．73％,t=7．131,P〈0．001;11．83％&#177;2．37％vs29．71&#177;3．47％,t=4．302,P〈0．001）,但体外生产的胚胎PCR阳性率比鲜精组显著提高（45．45％&#177;10．87％VS24．44％&#177;6．06％,t=1．750,P=0．013）。超微结构观察和双荧光探针检测都发现冷冻-解冻精子质膜完整性降低（8．34％&#177;4．21％VS65．67％&#177;6．46％,t=12．492,P〈0．001）,SCGE显示冷冻极显著增加了精子彗尾长度和彗星细胞比例（42．67μm&#177;4．56μmvs21．14／Lm&#177;2．36μm,t=5．644,P=0．005;60．00％&#177;4．00％vs17．37％&#177;2．57％;t=15．787,P〈0．001）。冷冻-解冻可以提高山羊精子转染外源DNA的效率,冷冻破坏精子质膜完整性,解除质膜的阻碍作用,是提高外源DNA转染效率的一个主要原因[动物学报54（6）：1089-1097,2008]。     

Strain Differences in Superovulatory Response, Embryo Development and Efficiency of Transgenic Rat Production

The differences between rat strains in superovulation response, in vitro and in vivo development of preimplantation embryos and overall transgenic efficiency was studied. The protocols for induction of superovulation using single injections of pregnant mare’s serum gonadotropin (PMSG) or minipumps with follicle stimulating hormone (FSH) were compared in Lewis (LEW), Wistar-Kyoto (WKY), and stroke-prone spontaneously hypertensive rats (SHRSP) or Sprague–Dawley (SD) and Wistar rats as representative inbred or outbred strains, respectively. The percentage of mated animals with positive superovulatory response was similar in all strains (60.0–100%). The mean number of ova per donor was not dependent on the kind of hormonal treatment used within each rat strain. In general, females from outbred SD and Wistar rats were more responsive to hormonal treatments than animals from inbred rat strains. In addition, SD female rats produced a significantly higher number of embryos per female in response to PMSG-treatment compared to all other strains. Between the inbred strains, SHRSP was the most effective for superovulation. In vitro development of intact zygotes to the blastocyst stage was not different between SD, Wistar and SHRSP rats. In contrast, in vitro development of WKY zygotes was significantly less efficient than in other strains. However, 2-cell stage embryos in vivo produced from SD, SD × Wistar and WKY animals showed no difference in competence to develop to blastocyst stage in vitro. The proportion of offspring developing after oviduct transfer of intact zygotes was similar in all strains (44.0–56.4%) with the exception of WKY rats (35.9%). We also compared the survival rate after injection, ability of manipulated zygotes to develop to term and overall transgenic efficiency in various rat strains. SD and SHRSP zygotes survived after microinjection better than the WKY and Lewis zygotes. No differences were found in the efficiency of transgene integration per newborn in different strains ranging from 5.7 to 16.7%. The results of this study demonstrate that different rat strains have varying responses to superovulation, sensitivity to microinjection, capability to develop in vitro until blastocyst stage or in vivo to term after transfer to foster mothers. Despite these differences all studied strains can be used for efficient transgenic rat production.     

PCR扩增同源重组片段筛选转基因胚胎

使用小鼠乳清酸蛋白基因（ＷＡＰ）启动子控制下的人集落刺激因子（Ｇ－ＣＳＦ）基因为显微注射片段,采用ＰＣＲ方法检测了转基因胚,为消除ＰＣＲ扩增中的假阳性结果,构建了两个具有部分同源性的亚克隆片段进行共注射．ＰＣＲ扩增片段跨越这一同源区域,仅当注射的片段能够整合并发生正常重组,转基因整合胚才能以相对高的比例扩增出特异性片段．结果表明,１、２和８细胞期的阳性率分别为１１．１％、５５．５％和４４．４％,较常规ＰＣＲ检测获得更为明确的结论,为在大动物转基因胚胎检测提供了依据     

Increased Efficiency of Transgenic Livestock Production

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.     

水牛精子蛋白质组双向电泳体系的建立和优化

建立和优化一种适合水牛精子蛋白质组学研究的双向电泳技术。以水牛精子为研究对象,比较两种不同配方的裂解液,以及不同上样量对其2-DE图谱质量的影响。结果显示,以7 mol/L尿素、2 mol/L硫脲、4%CHAPS、1%DTT、0.5%Cocktail of protease inhibitors为裂解液,24 cm胶条上样量200μg时,可获得较好的精子总蛋白质2-DE图谱。运用ImageMaster 2-Dplatinum分析软件检测出约500个蛋白质点,蛋白质大部分分布在等电点5-7之间,分子量范围约40-90 kD。     

高畸形率水牛精子与正常水牛精子差异表达蛋白的分离和鉴定

旨在分析高畸形率和正常水牛精子的差异表达蛋白.运用双向凝胶电泳以及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS/MS)分析鉴定出高畸形率和正常的水牛精子的差异表达蛋白,并对部分蛋白进行生物信息学分析.结果显示,高畸形率和正常水牛精子之间存在16个表达差异明显的蛋白点,与正常水牛精子相比,5个蛋白斑点表达量上调,6个蛋白斑点下调,3个蛋白斑点缺失,2个蛋白斑点在畸形率高的水牛精子特有.质谱鉴定16个差异蛋白,成功鉴定出6个差异蛋白斑点,对应4种蛋白:左旋天冬酰胺酶、热应激蛋白β-9、半乳糖激酶、β-微管蛋白-2C.研究表明,高畸形率和正常水牛精子蛋白质表达存在一定的差异.     

Strain-Dependent Differences in the Efficiency of Transgenic Mouse Production

Transgenic mouse production via pronuclear microinjection is a complex process consisting of a number of sequential steps. Many different factors contribute to the effectiveness of each step and thus influence the overall efficiency of transgenic mouse production. The response of egg donor females to superovulation, the fertilization rate, egg survival after injection, ability of manipulated embryos to implant and develop to term, and concentration and purity of the injected DNA all contribute to transgenic production efficiency. We evaluated and compared the efficiency of transgenic mouse production using four different egg donor mouse strains: B6D2/F1 hybrids, Swiss Webster (SW) outbred, and inbred FVB/N and C57BL/6. The data included experiments involving 350 DNA transgene constructs performed by a high capacity core transgenic mouse facility. Significant influences of particular genetic backgrounds on the efficiency of different steps of the production process were found. Except for egg production, FVB/N mice consistently produced the highest efficiency of transgenic mouse production at each step of the process. B6D2/F2 hybrid eggs are also quite efficient, but lyze more frequently than FVB/N eggs after DNA microinjection. SW eggs on the other hand block at the 1-cell stage more often than eggs from the other strains. Finally, using C57BL/6 eggs the main limiting factor is that the fetuses derived from injected eggs do not develop to term as often as the other strains. Based on our studies, the procedure for transgenic mouse production can be modified for each egg donor strain in order to overcome any deficiencies, and thus to increase the overall efficiency of transgenic mouse production.     

采用高分子介导精子作载体制备转基因泥鳅

为探讨树形高分子介导精子载体技术产生转基因动物,将泥鳅精子与具有标记基因LacZ的pCH110重组质粒和树形高分子在保存液内孵育,经DNA原位杂交检测发现树形高分子介导下精子携事外源DNA的效率得到较大的提高,将捕获了外源DNA的精子,再与泥鳅卵进行体外人工受精。由此发育的鱼苗经PCR和LacZ组织化学检测,获得了高比例的转基因泥鳅,外源基因LacZ在泥鳅幼苗头部得到了明显表达。     

Stem Cell Conditioned Media Contains Important Growth Factors and Improves In Vitro Buffalo Embryo Production

The present study was designed to investigate the effect of MSCs-conditioned media (CM) on quality buffalo embryo production in vitro. MSCs were harvested from Wharton’s jelly of 2-3 month old fetus and MSCs CM was collected. Immunocytochemistry and western blot assay revealed that MSCs secrete several important growth factors viz. FGF-2, IGF-1, LIF, TGF-β, and VEGF. Slaughterhouse derived culture grade cumulus oocyte complexes (COCs) were matured and fertilized in vitro. Presumptive zygotes were divided in four groups and cultured in vitro in respective media viz. group I (100% mSOF), Group II (100% Knockout Media DMEM+SR), Group III (50% CM + 50% mSOF), and group IV (100% CM). It was found that though the cleavage rate did not changed significantly (p < 0.05), but blastocyst rate was increased significantly (p < 0.05) in Group III and IV (24.24 ± 1.34 and 23.29 ± 1.25, respectively) compared to group I and II (16.04 ± 1.46 and 17.72 ± 0.94, respectively). Similarly, TCN was significantly (p < 0.05) higher in 50% CM and 100% CM replacement group (93.33 ± 1.91 and 92.13 ± 1.04, respectively) than the other two groups. It can be concluded from the study that MSCs secrete several important growth factors and MSCs-CM can be effectively used for enhancement of quality buffalo embryo production in vitro.     

通过双原核显微注射提高转基因小鼠研制效率的实验研究

目的建立高效的转基因小鼠制备技术,为开展遗传工程动物模型研究奠定技术基础。方法通过向小鼠受精卵原核中注入不同浓度的DNA分子,筛选最适注射用DNA浓度;将K14/hCTLA4-Ig基因表达载体分子通过显微注射分别导入小鼠受精卵雌、雄原核,并设立单原核注射对照组;利用输卵管腹壶部穿刺移植法将注射后的小鼠受精卵移植于同期发情的受体母鼠;利用PCR对出生的转基因首建小鼠进行筛选。结果最适DNA分子浓度为10ng/μl;在单、双原核注射组胚胎2细胞卵裂率分别为52.3%(132/253)和45.0%(108/240),差异有显著性(P<0.05);注射胚胎移植后体内存活率分别为18.1%(24/132)和16.7%(18/108),差异无显著性;转基因首建小鼠阳性率分别为3/24和5/18,转基因阳性小鼠占总注射胚胎的比例为1.2%(3/253)和2.08%(5/240),差异有极显著性(P<0.01)。结论尽管双原核注射对胚胎的2细胞卵裂率有一定影响,但通过双原核注射可有效提高转基因小鼠的制备效率。     

Transgenic Quail Production by Microinjection of Lentiviral Vector into the Early Embryo Blood Vessels

Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP) transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13–15 (HH13–15). A total of 80 embryos were injected and 48 G0 chimeras (60%) were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR), indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%). We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1%) were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7%) were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13–15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection.