液体培养过程中金花茶体细胞胚增殖优化

为获得大量性状一致的金花茶体细胞胚,试验采用单因素与完全随机设计,在液体培养过程中先后研究了碳源组合、脯氨酸浓度及培养基体积与接种量对其增殖的影响。结果表明：碳源组合对体细胞胚增殖无显著影响,但以40 g·L-1蔗糖与20 g·L-1山梨醇作为碳源时,低畸形胚的产生频率较低;脯氨酸浓度对体细胞胚增殖有极显著影响且适宜的浓度为2g·L-1;培养基体积、接种量及两者的交互作用均对体细胞胚增殖有极显著影响且其中影响最大的为培养基体积,最佳的培养基体积为45 mL,最佳的接种量为0.5或1.0 g。采用以上优化结果对体细胞胚进行液体培养,其增殖系数可达5.65且一致性良好。     

利用脂质体法建立转Toll样受体4基因大尾寒羊胎儿成纤维细胞株的研究

利用脂质体转染法获得转Toll样受体4(Toll-like receptors 4,TLR4)基因的成纤维细胞,将其作为供体细胞,再通过体细胞核移植技术构建重构胚,最终生产出转TLR4基因且性别可控的大尾寒羊。本研究通过原代培养大尾寒羊胎儿皮肤成纤维细胞,并进行SRY基因性别鉴定,利用脂质体转染法转入TLR4基因,然后分别从形态学与分子水平检测TLR4基因的表达,将稳定表达转TLR4的成纤维细胞核移植入绵羊去核卵母细胞中构建重构胚。最终获得5个稳定表达TLR4的阳性细胞克隆,经SRY基因性别鉴定为雌性细胞株,通过实时荧光定量PCR分析,在第4代转染的细胞中TLR4表达量最高,比未转染细胞高出7.4816倍(P<0.01),卵裂的重构胚中有EGFP的表达。试验成功构建出含TLR4基因的重构胚,为今后生产出性别可控的抗病转基因绵羊地方新品种奠定基础。     

抽提物处理供体细胞对克隆胚胎发育的影响

本研究采用爪蟾卵母细胞抽提物处理和牛胎儿成纤维细胞(JBCFF),并以发生重编程的细胞为核供体进行体细胞核移植,研究其对克隆效率的影响.分别超排3只爪蟾,收集卵母细胞后提取其抽提物,用BCA蛋白浓度测定试剂盒确定其蛋白含量,SDS-PAGE分析其中所含蛋白的种类.应用细胞膜透化剂Digitonin对建立的JBCFF进行通透处理和PI染色,筛选最适浓度;并用获得的抽提物处理牛体细胞,获得重编程细胞.以发生重编程的体细胞为供体进行核移植,同时比较离子霉素+6-DMAP和A23187+6-DMAP两种组合激活后克隆胚的体外发育能力.结果,3个爪蟾卵母细胞抽提物样品的蛋白浓度分别为56.2255、64.6570和71.2158 μg/mL,其中所含蛋白种类一致,主要集中在40~55、70~100 ku之间.经过连续的筛选,透化剂Digitonin对JBCFF通透处理的最适浓度为7 μg/mL,PI染色显示透化效率为55.44%.爪蟾卵母细胞抽提物与体细胞共孵育后继续培养6~7 d,细胞聚集形成"克隆簇".分别以"克隆簇"细胞和未处理细胞为核供体,制备的重构胚在融合率(92.83%和96.04%)、卵裂率(89.64%和89.78%)和囊胚率(24.06%和23.12%)均无显著差异(P>0.05);离子霉素+6-DMAP和A23187+6-DMAP两种激活方式对克隆胚胎的分裂率(92.16%和92.28%)和囊胚率(23.21%和24.18%)均无显著影响(P>0.05).爪蟾卵母细胞抽提物诱导和牛体细胞发生重编程,并恢复到较低的分化状态,但没有显著促进克隆胚胎的体外发育,可见缺少对胚胎发育起重要作用的重编程过程,还需要继续深入研究.     

酸枣成熟胚离体培养研究

为了建立酸枣成熟胚离体培养再生体系,以酸枣成熟胚为试验材料,研究启动培养基和增殖培养基对不同贮藏处理酸枣成熟胚萌芽率及增殖的影响。结果表明：以常温保存1a的成熟胚萌芽率较高（57.78％）,最佳启动培养基为不添加任何植物激素的M S培养基,而最适增殖培养基为1/2MS＋1.0 mg/L 6 BA＋0.3 mg/L NAA＋1.5 g/L PVP。     

不同生长素类型及ABA搭配对小麦幼胚再生效果的影响

生长素是小麦幼胚产生胚性愈伤组织的关键成分,培养基中适宜的生长素种类、浓度和组合决定着小麦幼胚再生效果。本研究以小麦基因型CB037幼胚为材料,在分别确定dicamba、2,4,5-T和picloram的适宜用量的基础上,比较了2,4-D、dicamba、2,4,5-T、picloram在适宜浓度下诱导再生的效果,筛选...     

Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: The effects of different in vitro oocyte maturation systems

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non‐defined (TCM‐199 + pig follicular fluid) or in a defined base medium (POM + epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7 days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.     

Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off‐target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off‐target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non‐specific cleavage of off‐target sites.     

湿地松组培继代增殖的影响因子

以湿地松抗病家系和非抗病家系的成熟合子胚为外植体,以不同的基本培养基成分、不同的激素成分及浓度配比和不同的培养方式,研究湿地松离体培养继代增殖的关键技术及主要影响因子。     

数字差异显示法在猪早期胚胎基因表达分析上的应用

借助于NIH生物信息中心Unigene里的数字差异显示软件平台,分析了猪植入前后早期胚胎发育9个EST文库的105 227个ESTs,筛选出了104个差异表达ESTs,对已知功能的差异表达基因进行GO分类分析,发现这些差异基因参与了广泛的生物功能和过程。同时,这些差异表达ESTs的聚类分析结果显示,体外和体内发育的囊胚基因表达存在较大的差异。对这些差异表达ESTs表达模式的分析结果显示,猪植入前早期胚胎发育阶段基因呈振荡模式表达,其中线粒体和核糖体相关基因的表达在囊胚中的表达丰度要明显高于其他阶段。表达数字差异显示的结果能为猪早期胚胎发育机理的研究提供线索。     

孵化期间鸡胚蛋内蛋白质转移的研究

对不同日龄的广西黄鸡胚蛋的卵白、卵黄,胚胎中粗蛋白,尿囊液中的尿素氮含量进行了测定,结果表明,这些指标呈一定规律性变化:前期卵黄、卵白中蛋白质含量稳定,卵白中蛋白质在孵化的第9和12天胚龄含量较高,之后极显著下降。卵黄中的粗蛋白质含量呈逐渐增加的趋势。胚胎中的粗蛋白含量从9～12d胚龄显著降低;尿囊液中尿素氮的含量在12～21d胚龄迅速增加,21d胚龄达到最高水平。说明蛋白在中期转移最多,后期利用率较低。