外源视黄酸对小鼠早期胚胎形态及Otx2基因表达的影响

以全反式视黄酸（ａｌｌｔｒａｎｓ－ｒｅｔｉｎｏｉｃａｃｉｄ，ＲＡ）体外处理ＥＢ（ｅａｒｌｙａｌｌａｎｔｏｉｃｂｕｄ）期和ＬＢ（Ｌａｔｅａｌｌａｎｔｏｉｃａｃｉｄ）期的小鼠胚胎，然后用洋地黄毒苷（ｄｉｇｏｘｉｇｅｎｉｎ）标记的Ｏｔｘ２反意义ＲＮＡ探针对整体胚胎进行原位杂交．０．５×１０－６ｍｏｌ～２×１０－６ｍｏｌ的ＲＡ处理ＥＢ和ＬＢ期胚胎１２ｈ后对其形态学和Ｏｔｘ２表达都没有影响；以４×１０－６ｍｏｌ的ＲＡ处理，抑制或减少了胚胎的前肠形成，经处理的胚胎的头褶没有对照的那样明显向腹面突出，其神经沟也不整齐；以４×１０－６ｍｏｌ的ＲＡ处理ＥＢ期的胚胎，其ＯｔＸ２表达范围剧烈地向前退缩或只有很微弱的表达；但以同样浓度处理ＬＢ期胚胎时，与对照胚胎相比，ＯｔＸ２的表达除了在极少数胚胎中变得弱一些外，在绝大多数胚胎中其表达模式没有变化．     

小鼠胚胎移植技术优化及对巨细胞病毒净化研究

目的 建立小鼠巨细胞病毒净化方法,以获得无MCMV感染的小鼠。方法 利用胚胎移植技术,通过使用不同激素、不同发情周期注射激素、受体鼠品系、不同的移植方法、不同时期胚胎移植,以及移植胚胎数量等对比试验,优化了净化条件。利用优化的胚胎移植净化方法,对供体鼠进行了净化。结果 SIGMA生产的激素,在10 IU剂量下超排得到的可用胚胎数量约为17枚/只;在小鼠发情间期超排得到的可用胚胎最多,超排的可用胚胎数约为23枚/只;选取C57雄性小鼠与ICR雌性小鼠交配的子一代作为受体鼠,产仔率达44.5%;输卵管移植较子宫移植效果好,产仔率为40.64%;移植2细胞胚胎的妊娠率为80%,明显优于单细胞和8细胞;受体鼠移植24枚胚胎时,产仔率达到了43.75%。利用优化的小鼠巨细胞病毒胚胎移植净化方法,净化得到了无MCMV感染的小鼠。结论 建立了小鼠巨细胞病毒净化方法,为获得无MCMV感染的小鼠种群提供了可靠的保障。     

Combinational electroporation and transplantation approach to studying gene functions in avian embryos

Gene transfection is an indispensable approach for studying gene function since it provides important information on gain- and/or loss-of-function. Chick embryos are also extensively employed for studying bio- logical function since they are easily accessible and can be maintained alive after manipulation. The combination of both techniques presents a powerful approach to under- standing how genes regulate embryo development. Fur- thermore, combining these approaches with tissue transplant techniques make even more attractive for elu- cidate gene function. Electroporation, employing parallelly fashioned electrodes, has been widely used in chick embryos. However, experimenters have been frustrated by unsuccessfully transfection in some embryonic tissue of interest because the electrodes were improperly positioned.We presently demonstrated the different patterns of orga- nizing and positioning the electrodes, in combination with tissue transplantation, to efficiently and specifically trans- fect the chick embryonic head, trunk neural tube, heart tube, somites and neural crest cells with the GFP reporter gene.     

伽马辐照对斑马鱼胚胎谷胱甘肽还原酶活性及基因表达的影响

谷胱甘肽还原酶(GR)是一种重要的抗氧化酶,其在生物体的抗氧化系统中起着关键作用.为了研究伽马辐照对斑马鱼胚胎GR活性及基因表达的影响,本实验用不同的辐照剂量(0.01、0.05、0.1、0.5和1 Gy)分别对三个发育时期(原肠胚期、体节期和咽囊期)的斑马鱼胚胎进行了辐照处理.分析了各组GR的活性,并采用荧光实时定量PCR技术(qRT-PCR)检测了GR基因的表达水平.结果显示,伽马辐照以剂量依赖的方式调整了斑马鱼胚胎GR的活性及GR基因的表达.随着辐照剂量的增加,GR活性及其mRNA的相对表达量呈现先升高后回落的趋势.这表明,GR是一种可以对伽马辐照作出早期预警的生物标志物.斑马鱼胚胎可以通过上调GR基因的表达,来增加GR的合成,从而提高对伽马射线的防御能力.但是,这种反应和调整是有限度的.咽囊期胚胎比其它两个时期的胚胎对伽马射线的防御能力更强.     

大獭蛤的胚胎、幼虫及稚贝的形态发育

在广西海洋研究所海滨公园育苗场进行大獭蛤(Lutraria maxima Jonas)的胚胎发育观察及人工育苗实验。实验亲贝取自北海市侨港码头。通过人工授精获取受精卵。取部分受精卵在实验室用显微镜进行胚胎发育观察及拍照，在囊胚期之前每隔10min取样1次，囊胚期之后每个半小时取样1次，胚胎发育观察至D形幼虫期。大部分受精卵在育苗池中进行人工育苗实验，育苗期间每天取样1次，在显微镜下进行观察、测量及拍照，并记录贝苗大小及发育期情况。实验期间水温变化为25．0～28．7℃，盐度为29．3‰～31．9‰。根据观察记录结果，得出大獭蛤胚胎发育及贝苗生长发育图谱，并描述了大獭蛤的胚胎发育及其幼虫、稚贝的形态。     

Effects of free sterols on the fluidity of polar lipids in rice dry embryo

Free sterols from rice dry embryo reduce the onset phase transition temperature and widen the transition range of total polar lipids and phosphatidylglycerol, which increases the membrane fluidity below their phase transition temperature and decreases the fluidity of membranes above their phase transition temperature. And 50% of free sterols result in a complete disappearance of phase transition. The fluidity of other main components of polar lipids is decreased by free sterols above 0℃. The results confirm that free sterols can modulate membrane lipid fluidity.     

Effect of monoclonal antibody to bull sperm sp18 on murine fertilization and embryos development

Western blot analysis revealed that one IgG1 monoclonal antibody (mAb) to sp18 family membrane proteins (Mr. 14, 16 and 18 ku) of bovine sperm reacted faintly with protein bands of 14, 18, 22, 30 and 60 ku (reducing) in samples of mouse sperm. The mAb also reacted to protein of egg lysozyme. Using a laser confocal microscope, indirect immunofluorescence (IIP) showed that the sp18 antigens were present in the posterior head of murine sperm. In murine in vitro fertilization (IVF) and embryo development trails, a total of 426 oocytes from C57BL/6 and F1 hybrid strain (CD1 × C57BL/6 cross) of 12 female mice were used in 3 independent trails. After preincubating capacitated sperm with 182 μg/mL of sp18 mAb in the modified TYH IVF medium for 15-20 min, cumulus-oocyte-complexes were introduced. The fertilization rate in sp18 mAb groups was 77.1 %, which was not significantly (P > 0.05) different from the nonspecific mouse IgG (79.2%) and non-IgG (80.3 %) control groups. Fertilized oocytes had been continuously cultured in modifed CZB medium. 100%, 100% and 97.9% of 1-cell embryos developed to 2-cell stage in sp18 mAb, nonspecific mouse IgG and non-IgG group 30 h after the start of fertilization, respectively. In the nonspecific mouse IgG and non-IgG groups, 64.1 % and 64.3% of embryos developed to the 4-cell stage, respectively, but all developing eggs in sp18 mAb groups arrested development in vitro at 2-cell stage. After zonae of 2-cell blocked embryos were enzy-matically removed with 0.5% pronase, detection of sp18 antigens by IIF indicated that the fluorescence scattered on two embryonic cells. For embryos fertilized in vivo and co-cultured with 182 μg/mL sp18 mAb, the numbers of 1-cell embryos reaching the 2-cell and 4-cell stage were 95. 2% and 70. 5%, which were not significantly (P>0.05) different from the control group (92.9% and 77.9%). These results indicate that the sp18 antigens on posterior head of mouse sperm were incorporated into the egg plasma membrane during fertilization, and played an active role in development of murine preimplanta-tion embryo.     

Analysis for the dorsalization potency of the animal blastomeres of the 16 cell stageXenopus embryo

The dorso-ventral axis ofXenopus embryo is established after fertilization. Blastula stage blastomeres acquire different identities as they have inherited different maternal materials which distribute radial symmetrically along the animal vegetal aixs in the full-grown oocyte, and are rearranged by the cortical rotation triggered by fertilization. The vegetal blastmeres demonstrate the different dorsalization potencies in the previous transplantation experiments. The data of blastomere explanting and RT-PCR analyzing indicate that the dorsal ventral bias also exists among the animal blastomeres even during the early blastula stage.     

利用CRISPR-Cas9 构建syncytinA 条件敲除小鼠

摘要: 目的构建syncytinA( 合胞素A) 条件敲除小鼠,为进一步研究syncytinA 在胎盘形成过程中发挥的融合及非融合作用及研究子痫前期病理模型提供基础。方法在用ES 细胞打靶完成syncytinA 外显子上游loxp 同源重组基础上,利用CRISPR-Cas9 得到syncytinA-loxp 小鼠。构建syncytinA-loxp 转基因载体及sgRNA,通过原核显微注射方法将构建好的doner、Cas9 及sgRNA 一并注射到C57 小鼠受精卵中,并移植入同期受孕代孕受体ICR 输卵管中获得子代小鼠。用PCR 方法检测子代鼠尾基因型, loxp 阳性小鼠与WT 交配获得syncytinA-loxp 小鼠。为了检测syncytinA-loxp 能否被敲除,通过与prime1cre 及zp3cre 交配获得syncytinA － / － ,PCR 及q-PCR 检测syncytinA 是否被敲掉。结果经PCR 及q-PCR 方法检测,我们成功得到syncytinA － / － 胚胎。结论syncytinA 条件敲除小鼠构建成功,为更好的研究它在胎盘及子痫前期中的功能提供了基础。     

DAP 玻璃化冷冻液对小鼠2-细胞胚胎的冷冻保存

摘要: 目的探索和优化小鼠2-细胞胚胎冷冻效果,建立小鼠胚胎冷冻保存技术以及超低温保存模型动物胚胎实验室所需的相关技术。方法选择3 个品系( C57BL/6、ApoE － / －、NOS3 － / － ) SPF 级4 周龄雌性小鼠,运用超数排卵、体外受精、玻璃化冷冻法,以及二细胞期胚胎复苏、体外培养等技术方法,观察上述实验效果。结果C57BL/6、ApoE － / －、NOS3 － / － 3 个品系小鼠超数排卵平均值分别为42. 98 个/只、27. 93 个/只、23. 11 个/只,体外受精2-细胞期胚胎率分别为68. 79%、32. 70%、23. 08 %,胚胎冷冻复苏后胚胎回收率72. 77%、80. 87%、83. 33%,存活率分别为56. 92%、54. 84%、70%,存活胚胎体外培养发育到囊胚比率分别为72. 37%、60. 78%、25%。结论选择4 周龄小鼠,按预定相同的技术方法做超数排卵、体外受精、胚胎冻存、胚胎复苏,呈现出较好效果,C57BL/6 小鼠优于基因工程小鼠,基本建立起小鼠2-细胞胚胎冷冻保存技术。