Expression Regulation Of Porcine HOXA10Gene And Establishment Of Transgenic Animal Model Of Hormone-Inducible Promoter

Improvement of litter size can bring huge economic benefits to pig industry, identification of crucial candidate genes and new approached affecting litter size are very important in pig research..Transgenic technology have a very good prospect in porcine reproductive traits improvement. In a previous study, Homeobox A10(HOXA10) was shown to be one of the most promising candidate genes which play major roles in endometrial differentiation and development, establishing the conditions required for implantation and normal pregnancy maintenance. HOXA10is a well-known transcriptional factor that can be regulated by estrogen (E2) and progesterone (P4). However, little work has focused on the regulation of the porcine HOXA10gene. Considering the important effects of HOXA10on embryo implantation, we investigated its expression and regulation mechanism, also provide theoretical basis and animal model for the improvement of porcine litter size.The results are as follows:1. The full-length of coding region of porcine HOXA10was cloned (1363bp), and its genomic structure were defined. HOXA10contains1exons and1intron. The cDNA of porcine HOXA10was2628bp in length, with a CDS of1236bp encoding a peptide of411amino acid residues, which showed90%and95%sequence similarity to that of human and mouse homologues, respectively.2. Analysis of the DNA sequences of HOXA10revealed2SNPs, one of them is in exon1. Allele frequency of this HOXA10MslI polymorphism revealed great variation between6porcine breeds. Association analyses showed that the MslI polymorphism had significant (P<0.05) association with total number born (TNB), and extremely significant (P<0.01) association with number born alive (NBA) and birth weight (WB);3. RT-PCR confirmed that the porcine HOXA10gene is highly expressed in the endometrium, bladder and kidney. QPCR showed that the expression of HOXA10was significantly higher on day15of gestation (gd15) in comparison to gd26(P<0.01), gd50(P<0.01) and day15of the estrous cycle (ed15) in the endometrium of Meishan pigs. In contrast, the highest expression in the endometrium of Yorkshire pigs was on gd50. Moreover, the abundance of HOXA10mRNA was the highest on gd15in Meishan pigs than in any other stage tested in the two breeds; 4. QPCR results showed that the endometrial expression of HOXA10is increased when the pregnant mice were injected progesterone (2mg/each per day), and the number of embryo is also increased, but the difference is not significant;5. Deletion analysis and reporter expression assays identified a promoter region (-1044bp to+18bp) that is responsible for E2and P4-induced HOXA10transcription. Moreover, this promoter region enhanced the E2/P4-induced HOXA10expression in Ishikawa cells.6. Lineralized pc3.1-Pro3-DNA plasmid was used to microinjection to establish transgenic mice and transgenic pigs. PCR and Southern blot analysis were used to identify the positive transgenic ones. Offspring carrying the foreign HOXA10were identified by PCR, and5of them were confirmed by Southern blot analysis. The two positive transgenic mice (F0) were mated to wild-type mice to establish transgenic lineages. PCR and Southern blot results showed there are9positive transgenic mice in F1, and21positive transgenic mice in F2. The positive transgenic rate was about40.9%and61.8%, respectively. RT-PCR analysis of F2showed that the foreign HOXA10were expressed in endometrium, kidney and bladder of all the ten female lineages. PCR analysis of F1and F2showed that the foreign HOXA10in the2positive Fo mice could be transmitted stably.In addition, QPCR results showed that the endometrial expression of HOXA10is increased significantly in pregnant transgenic mice on d5of gestation compared to the non-pregnant transgenic mice on d5of estrous cycle. Mean while the number of embryo is also increased.PCR and Southern blotting analysis identified4positive transgenic pigs which carried the foreign HOXA10gene, the positive rate is12.1%.In conclusion, we identified an E2/P4response element of the porcine HOXA10gene for the first time. Moreover, we assume that the porcine HOXA10gene may affect litter size by regulating implantation. The data from the present study contribute to the mechanism by which the porcine HOXA10gene regulates embryo implantation and prolificacy traits. We also established the hormone-inducible transgenic mice and transgenic pig which provide the animal model for the improvement of little size traits.