【作者】 宋根娣；

【导师】 郑振宇；

【作者基本信息】 河南农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 为了研究携带卵子突变基因(ovum mutant或Om)的DDK雌鼠与其它近交系雄鼠交配时表现出极性不育现象,本试验以杂合致死胚胎(DDK♀x BALB/C♂)为主要研究对象,纯合正常胚胎(DDK♀x DDK♂)为对照组进行了研究。通过对正常交配后植入前期受精卵的采集、观察,发现了杂合胚胎从8细胞期之后开始发生凋亡现象；进一步利用半定量RT-PCR技术,比较候选基因在不同交配组合植入前期受精卵中表达情况,从分子水平上探索Om基因的作用机制。本研究由两部分组成：试验一不同交配组合小鼠植入前期受精卵的采集及观察1、试验用交配组合的建立Om基因导致小鼠胚胎致死现象的产生在于交配组合的严格性,(DDK♀x BALB/c♂)在Om基因作用下出现胚胎致死现象,而DDK品系本交交配得到F1小鼠胚胎能够正常发育,未出现任何发育的异常情况。因此,选择纯合作为对照组,其植入前期受精卵的发育情况及候选基因表达情况可作为正常胚胎的对照标准。2、植入前各时期受精卵的采集及观察选取60日龄小鼠,按照超排处理与正常对照将雌鼠分成两组,并分别按照雌雄比例1：1方式合笼交配。根据胚胎发育时间,镜检观察、采集不同时期受精卵。结果表明,超排处理DDK雌鼠产生的胚胎,大量处于2细胞期发育停滞。正常交配方式中杂合胚胎在8细胞期后发生凋亡现象,并确定为死亡细胞。采集形态学上发育正常的2细胞至8细胞期发育阶段胚胎各70-110枚。试验二利用半定量RT-PCR检测候选基因在不同交配组合小鼠植入前期受精卵中的表达1、候选基因的选择目前在小鼠早期胚胎发育过程中,主要表达的功能基因有原癌基因、生长因子、生长发育及凋亡基因、白细胞抑制因子等几大类,其中各个功能基因表达量变化引起不同的影响。因此,试验选取8个功能基因作为候选基因,其差异表达能够导致胚胎的非正常发育,并且与Om基因引发的胚胎致死现象在胚胎学及生理学上极为相似。所以,杂合胚胎与纯合胚胎在发育过程中候选基因片段的表达差异,可认为是直接导致胚胎死亡的因素。2、半定量RT-PCR检测通过半定量PCR检测比较8个候选基因在杂合胚胎与纯合胚胎2细胞期、4细胞期、8细胞期受精卵中mRNA的相对含量。结果表明,在两种交配组合中,候选基因的中部分基因表达量发生差异,H-ras, TGF-α, C-myc基因在4细胞期,8细胞期两种交配组合中表达量相同,而EGF-R,Bcl-2基因在4细胞,8细胞期检测到表达量差异显著；Mcl-1,EGF在三个时期内表达量差异显著,并且差异表达的候选基因在杂合组合中表达量明显高于纯合组合。因此,纯合组合中胚胎细胞早期发育后期出现的致死现象,可能是由于这些差异表达基因的作用直接或间接导致的。本试验通过对携带Om基因的DDK小鼠杂合胚胎植入前受精卵发育过程的观察及一系列在着床前期影响胚胎正常发育的功能基因表达量差异的研究,分析Om基因引发的胚胎致死过程,揭示了Om基因是通过一类影响胚胎发育的功能基因而间接导致小鼠胚胎致死现象。为进一步阐明卵子突变基因的作用机理,完善小鼠胚泡形成过程中分子调控的相关理论奠定了一定的基础。更多还原

【Abstract】 In order to study the mutant gene (ovum mutant,Om) of DDK females with other inbred strains showed mating male sterility phenomenon of polarity, that is, (DDK♀x alien♂) F1-generation in the developing embryo to pregnancy 3-5 day obstacles due to the death of the blastocyst formation process. In this study, carried out by lethal hybrid embryos (DDK♀x BALB/C♂) as the main study, while the use of normal embryos of the cross (DDK♀x DDK♂) for control of the research work. Through the normal pre-implantation zygotes in mating collection observation, found that the death of hybrid embryos from the 8 cell stage also after the beginning of morula apoptosis occurred; through semi-quantitative RT-PCR technology to compare expression of candidate genes in different combinations of mating pre-implantation fertilized egg.This study is divided into two parts:Test-ⅠCombination of different mating mice pre-implanted fertilized egg collection and observation1. Experiment with the establishment of mating combinations.Om gene in mice resulted embryonic death phenomenon is the strict combination of mating, (DDK♀x BALB/c♂) can be embryonic lethal gene that is the role of Om, and DDK strains obtained from the mating of the F1 cross to the normal development of mouse embryos, without any abnormal development of the situation. Therefore, the choice of the cross as a control, the pre-fertilized egg implants and the development of candidate gene expression in normal embryos can be used as reference standard.2. Pre-implantation of the fertilized eggs during the collection and observationSelect the 60-day-old mice, super-exclusive deal with the normal female female ratio of 1:1, respectively, in accordance with the way male and female mating cage together. According to embryonic development time, microscope observation, collecting eggs at different stages. The results show that DDK ultra-exclusive deal with a large number of female embryos at the 2-cell developmental stagnation and can not conduct a follow-up test. Mating the normal mode hybrid embryos died after 8-cell apoptosis, identified as dead cells. Acquisition and development morphology were normal on 2 cells to 8-cell embryos of various developmental stages 80-100 pieces.Test-ⅡSemi-quantitative RT-PCR detection expression of candidate genes in the different mating combinations in mice pre-implanted fertilized eggs.1. The choice of candidate genesCurrently in the process of early embryonic development in mice, the main function of gene expression of proto-oncogenes, growth factors, growth and apoptosis genes, such as leukocyte inhibitory factor categories, including various functional changes in gene expression caused by different effects. Therefore, the test to select differentially expressed due to their non-normal development of embryos and embryonic Om gene triggered by the phenomenon of death in the embryology and physiology is very similar to the eight functional genes as a candidate gene, hybrid-type embryo death settlement with the normal development of embryos in the process of of candidate differentially expressed gene fragment can be considered as a direct result of fetal death.2. Semi-quantitative RT-PCR detectionThrough semi-quantitative PCR detection comparison in eight candidate genes with the hybrid embryos to death to pay the normal 2-cell embryos,4 cell,8 cell stage fertilized eggs in the relative content of mRNA. The results showed that the test carried by the DDK mouse gene Om hybrid fertilized preimplantation embryos died during development of observation and the impact of a series of pre-implantation embryos in the normal development of the functional differences in gene expression studies, analysis of gene Om triggered by the process of embryonic death, Om revealed a class of genes is affected embryonic development and, indirectly, the functional genes in mouse embryos resulted in the phenomenon of death. To further clarify the role of egg mutation mechanism, and improve the process of mouse blastocyst formation of molecular control theory to lay a certain foundation.更多还原