Construction And Validation Of LongSAGE Library In Pre-Implantation And Research Of Differential Expression Gene In Mouse

The ultimate goal of all the creature in nature to survive is to extend their population, therefore, multiplying is the most important event to any creature. And the begining of life is concerned by human all the time, so that to carry out research on the field of reproduction is significant for human being. During the process of reproduction, embryo implantation is the key step to gestation. There are two terms for implantation:1. receptive endometrium and embryo invasing;2. the synchronization between the receptive endometrium and embryo invasing. To achieve these two points, it is needed to establish close links of material and information between the mother and embryo. Many regulatory factors are involved in the process, such as endocrine regulators , paracrine regulators and secretion regulators, and regulate accuratly to the endometrium and the embryo in terms of time and space through a variety of cell signal transduction. But the time of receptive endometrial to allow embryo implantation is very short, known as the " implantation window". Through taking off transparent, positioning, adhesion, invasion and implantation, embryo have completed the first step of pregnancy in implantation window. Because many genetic regulation and the complex network of regulatory factors have been involved, the detailed molecular mechanism of embryo implantation and early development has been unclear and has been the issue of reproductive research. Objective:By getting the genes related with embryo implantation, to establish experimental and theoretical basis for clarifying the mechanism of embryo implantation.Method:LongSAGE gene expression library on pregnant d3 had been constructed by the serial analysis of gene expression, compared with completing LongSAGE tag library on pregnant d5. The reliability of the library was verified with RT-PCR, situ hybridization, and unknown tags from the library were analyzed by 3'-RACE technique.Results:The total numbers of tags sequenced were 1697 for d3. There were 1222 tags expressed at d3, of which 625(51%)genes were single-matched on d3 respectively, and 305(25%) multiple matching tags, 293(24%) new tags.The results show that the expression level of 17 LongSAGE tags on pregnant d3 was significantly higher than that of pregnant d5 (more than 25 times). The expression level of 5 LongSAGE tags on pregnant d5 was significantly higher than that of pregnant d3 (more than 20 times). The expression levels of 3 new tags on pregnant d3 were significantly higher than that of pregnant d5(at least 12 times higher), Similarly, the expression levels of 2 new tags on pregnant d5 were significantly higher than that of pregnant d3(four to five times higher). The seven differential expression genes were verified by RT-PCR. For unknown 15 differential expression tags, we have obtained 3'-fragment Conclusion:1. In this study, we successftlly established a ?ongSAGE gene library on pregnant d3 in mouse, and obtaindd Pome tags of significant dafferential expression bq comparing to the LongSAGE gene library on pregnant d5,2. The rediability of the LongSAGE gene libbary was repified by RT-PCR.3. Some unknown tags from the LongSAGE gene library were analyzed by improving the 3'-RACE method and confirmed that the unknown tag TCATAACGATTAACCTG belongs to gi: 34538597.4. Function and role of the differential expression genes between pregnant d3 and d5 were discussed in early pregnancy. This is very valuable for elucidating implantation mechnism.