Construction Of Transgenic Mice Harboring Porcine Kiss1and Fshr Gene And Evaluation Reproductive Performance

Kissl and FSHR genes play a significant role in litter size, reproductive physiology in the field of livestock. Kisspeptin function appears to be critical for gonadotropin-releasing hormone (Gonadotropin Releasing Hormone, GnRH) secretion, and plays the role of the central node in the gonadal axis system, can directly act on GnRH neurons, stimulating the GnRH secretion, positively regulating of LH and FSH secretion during the estrous cycle. In female mammals, FSH plays main role in maintaining the reproductive function and normal gonadal development and promoting the growth of ovarian follicular granulosa cell proliferation and estrogen synthesis and secretion, moreover stimulation of follicular cells LH receptors. It plays an important role in regulating follicular growth and maturation, directly have effects on the quantity and quality of follicular development. FSHR is an indispensable factor in FSH physiological function. The objective of this study was to investigate the correlativity between two genes and reproduction by constructing eukaryotic expression vector pCaMKIIa-Kissl which was expressed in the hypothalamus of mice, while AMH-pFSHR was synthesized by Generay company which specific expressed on mouse granulosa cells. Then pCaMKIIa-Kissl and AMH-pFSHR linear fragments were microinjected into mouse zygotes to generate transgenic mouse. Genetically modified positive rate was verified by Southern blot technology; while the Genome Walking was used to refine chromosomal location for exogenous genes; and simultaneously transgenic mice and their offsprings little size were detected. Therefore confirmed that whether they can promote follicle development and maturation, which providing new strategies for improving little size. The main results are as follows,1. Porcine Kissl gene CDS was cloned by PCR and was inserted into pMD19-T vector. Restriction enzyme digestion and sequencing result confirmed that the recombinant vector was correctly constructed, while sequence alignment showed that the homology ratio was100%compared with GenBank. Therefore Kissl gene was inserted in pCaMKII vector, and the recombinant vector pCaMKⅡα-Kissl was verified right by Restriction enzyme digestion and sequencing result.2.In order to build transgenic mouse, the vector of pCaMKIIa-Kissl and AMH-pFSHR were linear treated and were microinjected into mouse zygote, respectively. We used PCR to detect the mouse pKissl positive ratio by extracting genomic DNA of71GO generation, the results showed that there are four positive individuals and the positive rate was5.63%(4/71). Subsequently, the positive ratio was4.22%(3/71) was identified by Southern blot technology. pFSHR of43GO generation mice were detected by PCR which resulted in nine positive individuals with positive rate of20.93%(7/43). The positive rate was6.97%(3/43), which was confirmed by Southern blot verification, ultimately.3. When using Genome Walking to acquire pKissl gene location, we found that one individual has two location sites. Genetically positive mice were preceded to Genome Walking analysis in order to locate3individuals of pFSHR gene location sites. Each individual has one insert site.4. The GO generation male mouse were mated with negative individuals to generate F1generation mice, the positive rate of detection was43.05%(65/151) by PCR test, ultimately. F1generation positive mice exhibited much litter traits. The F2generation positive rate of29.33%(22/75) were obtained by pFSHR gene. F1generation positive individuals selfed with negative littermates subsequently. We found that pFSHR mice and offsprings showed a trendency towards multi-litter size and high survival rate.