We have investigated the mechanism for embryo development block in vitro and to improve the development rate of golden hamster embryos in vitro. Intracytoplasmic sperm injection (ICSI) technique was used to produce golden hamster ICSI embryos. The changes in the histone acetylation and the expression of histone deacetylase and related genes were analyzed by immunocytochemical staining and real-time PCR both in golden hamster in vivo embryos and in ICSI embryos. Aged oocytes significantly increased the oocyte spontaneous activation rate. In vitro cultured ICSI embryos suffered from severe development block in M199TE medium. Expression of histone deacetylase 1 (HDAC1) was significantly decreased in the nuclei of the arrested ICSI 2-cell embryos, and its nuclear and cytoplasmic expression pattern was also markedly altered. The acetylation level of H4K5, however, was not significantly changed between golden hamster in vivo embryos and ICSI embryos. HSPA1A and MYC, the marker genes for zygotic genome activation (ZGA), were transcriptionally decreased in arrested ICSI 2-cell embryos. Transcription of HDAC1 was also downregulated in these embryos, whereas the mRNA expression of the proapoptotic gene, BAX, was not changed. These results indicate that the golden hamster ICSI embryo development block during ZGA is associated with decreased nuclear expression and altered expression of HDAC1. HSPA1A, MYC, and HDAC1 mRNA levels, which decrease, resulting in ZGA failure.

In order to investigate Kcnq1 gene function in golden hamsters,Kcnq1 gene was cloned and identified,and its expression differences was analysed in this study.Total RNA was extracted from golden hamster heart and primer TK1 was designed according to the rats Kcnq1 conservative sequence.Kcnq1 cDNA fragment was amplified from the heart using RT-PCR method.The purified cDNA was specifically linked with pMD18-T vector using the heart cDNA fragment as a template,which was then transformed into competent E.coli DH5α.After that,the recombinant was screened and identified and DNA sequencing.Total RNA was extracted from golden hamster heart,liver,spleen,lung and kidney,and Kcnq1 cDNA fragment was amplified using RT-PCR,expression differences in a variety of organs were detected by Real time PCR.The golden hamster Kcnq1 gene was cloned and total length was 477 bp,which indicated that it encoded 159 amino acids.The nucleotide and amino acid sequences had high homology when compared with rat etc..Real time PCR results showed that Kcnq1 gene was highest highly expressed in the heart,lung and kidney,less expressed in the spleen,almost no expressed in the liver.The expression in the heart was the highest.This study laid the foundation for further investigation for Kcnq1 gene function in golden hamsters.

Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEO(r) was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase-PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows.

V13KL is a kind of antimicrobial peptide,which is obtained by chemical synthesis. It is with characteristic of highly efficient, broad-spectrum and low hemolysis. First, V13KL encoding gene fragment was cloned into the pro- karyotic expression vector pET30b （＋）, and enterokinase restriction sites was designed at N-terminal. Then, pET30b（＋）-VK13L recombinant plasmid was transformed into E. coli expression strain BL21 （DE3）. The induction conditions were optimized, which make the peptide highly expressed. The identification by Tricine-SDS-PAGE and Western blotting indicated that the molecular weight of the protein was as expected. The results showed that V13KL gene was successfully cloned and expressed , and the protein expression could be detected in the induction system when 1 mmol/L IPTG was added up to 6 h.