Gene Function Analysis Of HOXA10 And Generation Of Transgenic Mice And Pigs

Reproductive trait is one of the most important economic traits in pig production, of which litter size is one of the major factors influencing porcine reproduction. Reducing the porcine embryo/fetus mortality, improving the survival rate of fetus, is the key to improve litter size. During embryo pre-implantation or implantation, the majority of embryonic death occurs. Thereby, embryo implantation is a critical period which can affect litter size. Studies have shown that the HOXA10(Homeobox A10) gene, as a well known transcription factor, was regulated by estrogen and progesterone, highly expressed in implantation window, and played a critical role in the establishment of embryonic implantation and maintenance of pregnancy. Our goal is to produce a transgenic animal, in which HOXA10 highly expressed in the specific tissue at the specific period via transgenic technology, reducing embryo mortality, increasing litter size.In this study, experimental data were accumulated from transgenic mouse model to lay the foundation for the preparation of high fecundity transgenic pigs; by transgenic cloning technology, it is desirable to obtain transgenic cloned pigs of high reproductive potential and get access to new research material; understanding the mechanism of HOXA10 gene in regulating the process of embryonic development and implantation, has important theoretical and practical significance for improving litter size in sows.The results are as follows:1. To study the effects of the porcine HOXA10 promoter fragment on the expression of the HOXA10 gene in vivo, we generated a transgenic mouse model using pronuclear microinjection. Two of the founder mice were identified as transgenic mice by Southern blotting and the full-length amplification of the transgene fragment. Q-PCR was used to examine HOXA10 expression in various tissues of transgenic mice, the results showed that HOXA10 was expressed in the kidney, urinary bladder and endometrium tissues, with low levels of expression in the heart, spleen and ovary. Treatment with progesterone and estradiol benzoate, the expression level of HOXA10 was significantly increased in transgenic mice compared with that of wild-type mice, and the expression of the HOXA10 downstream target genes ETA(Endothelin A) and Phgdh(phosphoglycerate dehydrogenase) were also changed. Furthermore, the litter size of transgenic females increased about 1 pup more than that of wild-type females for the second to later parities(P = 0.08), and we interpreted it as a tendency for transgenic HOXA10 expression to increase litter size.2. We have produced HOXA10 transgenic cloned pigs previously, and now obtain 56 offsprings, of which 25 pigs were detected positive, including 11 female. The HOXA10 expression of G0 transgenic cloned pigs was measured in 12 tissues. Result showed that the expression of HOXA10 gene was most abundant in the kidney, large intestine and urinary bladder, weak in the muscle and lymph node, absent in the heart, liver, spleen, lung, testis, fat and small intestine.3. We detected the physiological and biochemical parameters in 4 G0 transgenic boars and part of G1 pigs, and found the platelet numbers in the G0 and G1 of boar(both of the positive and negative) were lower than those of non-transgenic pigs and reported in the literature. In the biochemical parameters, the content of testosterone in G0 boars was lower than non-transgenic boars, and found no abnormalities in other parameters.4. Through the sex identification, liposome mediated transfection of pc3.1-PF3-HOXA10, G418 selection, monoclonal selection and PCR detection of the porcine fetal fibroblast cells, 4 strains of positive cell lines were established.5. Selecting one of the monoclonal cells with good condition as nuclear donor cells was to produce 3 HOXA10 transgenic cloned sows. Using Q-PCR and Genome Walking to detect the exogenous gene copy number and integration site, the results showed that the copy number in each pig was 27.70, 19.88, 25.26, respectively. The foreign fragment may be integrated in the chromosome 4, chromosome 9, chromosome 13, chromosome 14.6. m RNA and Lnc RNA expression profiles screening was performed by microarray when the HOXA10 gene expression level increased in Ishikawa cells. A total of 907 m RNAs and 1,026 Lnc RNAs were identified as differentially expressed transcripts between HOXA10 overexpressed and control Ishikawa cells. Among these, 339 m RNAs were identified to be upregulated and 568 m RNAs were downregulated(Fold Change ≥ 2, P < 0.05 and Q < 0.05). Further analysis of GO and KEGG pathway, the differentially expressed m RNAs were found to participate in various biological processes, such as the upregulated m RNA may participate in blood vessel development, cell adhesion, cell migration, etc. And the downregulated may be involved in cell cycle, intracellular transport, protein localization in nucleus, etc. From the data of KEGG pathway analysis, we found that the differentially expressed m RNAs mainly participated in Cell adhesion molecules, Cell cycle and so on.7. And 732 Lnc RNAs were upregulated, and 294 were downregulated(Fold Change ≥ 2, P < 0.05 and Q < 0.05) according tho microarray data. Of the Lnc RNA classification and subgroup analysis, 1,514 enhancer-like Lnc RNAs were detected in this study. And 43 of these enhancer-like Lnc RNAs were upregulated, 16 of them were downregulated. Of the 9,987 Linc RNAs(long intergenic noncoding RNAs) were identified by the present study, including 274 upregulated and 98 downregulated Linc RNAs. We integrated the differentially expressed Lnc RNAs and their adjacent differentially expressed m RNAs data, to predict the function of Lnc RNAs. Then we chose the m RNAs which involved in focal adhesion and blood vessel development to predict the Lnc RNAs which may relate with embryo implantation.