Timing and sequence requirements defined for embryonic maintenance of imprinted DNA methylation at Rasgrf1

Epigenetic programming is critical for normal development of mammalian embryos. Errors cause misexpression of genes and aberrant development (E. Li, C. Beard, and R. Jaenisch, Nature 366:362-365, 1993). Imprinted genes are important targets of epigenetic regulation, but little is known about how the epigenetic patterns are established in the parental germ lines and maintained in the embryo. Paternal allele-specific expression at the imprinted Rasgrf1 locus in mice is controlled by paternal allele-specific methylation at a differentially methylated domain (DMD). DMD methylation is in turn controlled by a direct repeat sequence immediately downstream of the DMD which is required for establishing Rasgrf1 methylation in the male germ line (B. J. Yoon et al., Nat. Genet. 30:92-96, 2002). To determine if these repeats have a role in methylation maintenance, we developed a conditional deletion of the repeat sequence in mice and showed that the repeats are also required during a narrow interval to maintain paternal methylation of Rasgrf1 in developing embryos. Removing the repeats upon fertilization caused a total loss of methylation by the morula stage, but by the epiblast stage, the repeats were completely dispensable for methylation maintenance. This developmental interval coincides with genome-wide demethylation and remethylation in mice which most imprinted genes resist. Our data show that the Rasgrf1 repeats serve at least two functions: first, to establish Rasgrf1 DNA methylation in the male germ line, and second, to resist global demethylation in the preimplantation embryo.     

High toxicity,low receptor density,and low integration frequency severely impede the use of adenovirus vectors for production of transgenic mice

Although many methods are available for introducing genes into the mammalian germ line, none is ideal for genetic manipulation of livestock or primates. These organisms produce relatively few offspring in each reproductive cycle and they have long generation times. For these reasons, a recent report that adenovirus vectors can efficiently insert genes into the mouse germ line by embryo infection is of considerable interest. Adenovirus vectors have a high cloning capacity, can be produced in high titers, and can infect a wide variety of cell types. We have investigated in more detail the potential for such vectors to infect embryos and integrate their DNA into the genome. We exposed mouse embryos to adenovirus vectors that express bacterial beta-galactosidase (LacZ), and studied expression in the preimplantation period, toxicity of the vectors, and the frequency with which fetuses and pups integrate vector DNA. Our findings indicate that fully functional adenovirus receptor does not appear until the two-cell stage of development. Successful infection is associated with high toxicity, such that viral titers must be balanced to achieve high infection with tolerable levels of toxicity. Screening of 94 animals after embryo infection revealed a single positive polymerase chain reaction signal, which is indicative of the presence of the lacZ gene. This finding could not be confirmed by Southern blotting, which indicates that the founder animal was a genetic mosaic for the exogenous DNA. We conclude from these experiments that adenovirus gene transfer vectors are not readily usable for germ line gene insertion.     

Twenty years of research on primordial germ cells.

Just twenty years ago I was preparing a research project centred on establishing methods for the isolation and culture of mouse primordial germ cells (PGCs). The project had been suggested to me by Anne McLaren and was to be developed at the Medical Research Council (MRC) "Mammalian Development Unit" in London under the direction of Anne herself. At that time I was a young postdoctoral researcher at the Institute of Histology and Embryology of the University of Rome "La Sapienza" and did not imagine that my decision to be involved in this project would signal a profound switch in my scientific life. From then on my research would mostly concentrate on primordial germ cell biology. I feel like saying that the modern history of mammalian primordial germ cells began twenty years ago at the MRC Mammalian Development Unit under Anne McLaren's impulse. It is not surprising that among the most active researchers in the last twenty years in studying mammalian primordial germ cells, three, namely Chris Wylie, Peter Donovan and myself, began their studies under Anne McLaren's guidance. Over the years, Anne's suggestions and encouragement were always precious for my studies and her presence marked my most important findings on PGC biology. She often invited me to present the results obtained in my laboratory to workshops and congresses. In the present article some of these results particularly influenced by Anne's teaching and suggestions will be briefly reviewed.     

单个植入前胚胎构建cDNA文库

以PCR为基础的单个植入前胚胎cDNA文库构建是一种快速、可重复和高效的建库方法。单个植入前胚胎cDNA文库作为一种重要的新兴基因资源库 ,为新基因的克隆和鉴定奠定了坚实的基础 ,不仅解决了胚胎研究材料受限的问题 ,而且在时间上更加精确 ,更符合胚胎发育的规律。是研究早期胚胎发育基因表达的一种有效手段。随着这一技术的不断改进和与其它技术的有机结合 ,必然为人们进一步揭示胚胎发育的分子机制开创一个崭新的局面。     

小鼠单个植入前胚胎SSH方法的可行性

为建立一种能够一次性分离任意两个植入前胚胎之间全部的差别表达的基因的方法 ,在已有的单胚胎操作技术的基础之上 ,对单个植入前胚胎抑制性消减杂交 (singlepreimplantationembryosuppressionsubtractivehybridization ,SPE SSH)方法进行了初步的探索 ,分离到OM2和MⅡ d 2的基因片段 ,经GenBank和文献检索发现 ,这两个基因具有在MⅡ期和 2细胞期特异性表达的特点 .利用cDNA阵列所进行的鉴定也获得了同样的结果 ,而且所使用的材料极少 ,说明SPE SSH是一种强有力的分离和识别早期发育相关基因片段的实验技术 .若与单个卵裂球分离技术相结合 ,还可用于分离和识别人类早期分子诊断的标记性基因     

Early human embryo metabolism

Non-invasive microanalytical methods have been devised to study the energy metabolism of single human preimplantation embryos. Psyruvate, which is added routinely to all media used to culture human embryos, is consumed throughout the preimplantation period, with glucose assuming an increasing role at embryo compaction and blastocyst formation. All of the glucose consumed may be accounted for by the appearance of lactate in the incubation medium. The enzyme hexokinase my be involved in regulating this aerobic glycolysis. There is cosiderable indirect evidence for the utilisation of endogenous as opposed to exogenous energy substrates, the most likely candidate being protein. Information on early human embryo metabolismis likely to find application in a number of areas: these include the improvement of techniques for assisted human conception, notably in the selection of embryos for transfer following In Vitro Fertilisation; the diagnosis of gentic defects at the preimplantation stage; increased undersding of the causes of implantation failure and miscarriage, and the development of novel post-coital contraceptives.     

兔移核重构胚再程序化相关基因片段的分离与鉴定

用单个植入前胚胎mRNA差别显示方法（Single Preimplantation Embryonic mRNA Differential Display Reverse Polymerase Reaction,SPEDDRTPCR）,以家兔移核重构发育至2细胞、8细胞时期的胚胎及囊胚作为起始材料。研究家兔移核重构胚再程序化相关基因的表达。获得了25个与再程序化相关的基因片段,完成了对所有片段的克隆、序列分析,其中5个反向Northern证实的片段在从MⅡ到囊胚的发育阶段中呈现特异性表达的特点。这项研究为再程序化相关基因全长的分离以及功能研究奠定了良好的基础。     

兔单个植入前克隆胚胎cDNA文库的构建

人与小鼠和牛在正常胚胎植入前发育过程中基因活化的研究已经取得了长足的进展 ,但是还没有对同期克隆胚胎相关研究的报道 .利用单个家兔植入前移核重构胚胎成功地构建了MⅡ卵母细胞及发育至 4 、8 细胞期的胚胎和囊胚的特异性cDNA文库 .并用 β肌动蛋白和LAPTM4α证实这类文库是可靠的 .以 8 细胞期移核重构胚cDNA文库为例 ,随机挑取克隆进行测序分析 :其中 2 3的基因EST片段可以在GenBank或EST库中找到同源序列 ,约 1 3的EST片段属于未知的新片段 ,表明这是一类重要的新兴基因资源库 (期特异性EST库 ) .这种利用单胚胎构建cDNA文库的方法 ,解决了胚胎研究材料受限的问题 ,在时间上更加精确 ,更符合胚胎发育的规律 ,也能够更加准确地反映出一些克隆胚胎的异常表型 ,是研究早期胚胎发育基因表达以及克隆胚胎再程序化基因表达的一种有效手段 .     

Expression of beta-galactosidase in preimplantation ovine and porcine embryos

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

胚胎发育过程中的DNA甲基化作用

哺乳动物的正常发育取决于表观遗传学调控机制准确无误地运行.其中尤为重要的是发生在原生殖细胞和胚胎中的基因组范围内的DNA甲基化模式重排等表观遗传学修饰.胚胎发育过程中的DNA甲基化作用与基因印记的建立、基因表达的调控以及细胞和胚胎的形态建成都密切相关.DNA甲基化发生机制和功能的阐明将对哺乳动物个体发育与人类疾病研究有重要意义.