Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters.

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.

Functional analysis of the upstream regulatory region of chicken miR-17-92 cluster.

miR-17-92 cluster plays important roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis. The transcriptional regulation of miR-17-92 cluster has been extensively studied in mammals, but not in birds. To date, avian miR-17-92 cluster genomic structure has not been fully determined. The promoter location and sequence of miR-17-92 cluster have not been determined, due to the existence of a genomic gap sequence upstream of miR-17-92 cluster in all the birds whose genomes have been sequenced. In this study, genome walking was used to close the genomic gap upstream of chicken miR-17-92 cluster. In addition, bioinformatics analysis, reporter gene assay and truncation mutagenesis were used to investigate functional role of the genomic gap sequence. Genome walking analysis showed that the gap region was 1704 bp long, and its GC content was 80.11%. Bioinformatics analysis showed that in the gap region, there was a 200 bp conserved sequence among the tested 10 species (Gallus gallus, Homo sapiens, Pan troglodytes, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Possum, Danio rerio, Rana nigromaculata), which is core promoter region of mammalian miR-17-92 host gene (MIR17HG). Promoter luciferase reporter gene vector of the gap region was constructed and reporter assay was performed. The result showed that the promoter activity of pGL3-cMIR17HG (-4228/-2506) was 417 times than that of negative control (empty pGL3 basic vector), suggesting that chicken miR-17-92 cluster promoter exists in the gap region. To further gain insight into the promoter structure, two different truncations for the cloned gap sequence were generated by PCR. One had a truncation of 448 bp at the 5'-end and the other had a truncation of 894 bp at the 3'-end. Further reporter analysis showed that compared with the promoter activity of pGL3-cMIR17HG (-4228/-2506), the reporter activities of the 5'-end truncation and the 3'-end truncation were reduced by 19.82% and 60.14%, respectively. These data demonstrated that the important promoter region of chicken miR-17-92 cluster is located in the -3400/-2506 bp region. Our results lay the foundation for revealing the transcriptional regulatory mechanisms of chicken miR-17-92 cluster.

[Characterization of chicken PPARγ expression and its impact on adipocyte proliferation and differentiation].

To characterize the chicken PPARγ gene expression and its impact on chicken adipocyte proliferation and differentiation, western blotting approach was conducted to investigate the expression of PPARγ in various chicken tissues and the difference of expression level in abdominal adipose tissues between the NEAU broiler lines divergently selected for abdominal fat content. The expression of PPARγ gene was suppressed in chicken adipocytes using RNAi technology, and the roles of PPARγ gene in the adipocytes proliferation and differentiation were investigated by MTT assay and Oil Red O staining extraction assay, respectively. After PPARγ gene was downregulated, the expression level of other transcript factors and marker genes related to the adipocyte differentiation was detected by Real-time PCR and Western blotting analyses. The results showed that PPARγ highly expressed in abdominal adipose tissue, gizzard, spleen, kidney, lowly expressed in heart, and not expressed in liver, breast muscle, leg muscle, and duodenum. Meanwhile, PPARγ expressed much higher in fat birds than in lean ones in abdominal adipose tissue at 5 and 7 weeks of age (P<0.05). RNAi analysis showed that knockdown of PPARγ gene increased chicken adipocyte proliferation and decreased cell differentiation and significantly decreased the expression levels of C/EBPα, SREBP1, A-FABP, Perilipin1, LPL, and IGFBP-2 (P<0.05). In summary, PPARγ gene may be related to the broiler abdominal fat deposition, and be probably a key regulator of chicken adipocyte proliferation and differentiation.

[Chicken SREBP1 gene antiserums preparation and its tissue expression characterization].

AIM: The aim of this study was to prepare the antiserums against chicken sterol regulatory element binding protein1 (SREBP1), and to analyze the expression of SREBP1 in chicken tissues. METHODS: The nuclear import sequence of SREBP1 was analyzed using the DNAStar programs to predict its major antigen epitopes, the fragment coding for SREBP1 major antigen epitopes (832-1 302 bp) was amplified by RT-PCR and inserted into pGEX-4T-1 to construct the expression vector pGEX-4T/SREBP1. The recombinant GST/SREBP1 was expressed in E.coli with IPTG induction and purified by Glutathione Sepharose 4B affinity chromatography. The purified recombinant GST/SREBP1 was used as immunogen in rabbits, and the titer and specificity of SREBP1 antiserums were detected by ELISA and Western blot. The tissue expression of chicken SREBP1 was analyzed with the antiserums. RESULTS: The titer of the antiserum determined by ELISA was 1:102 400, and the antiserums exhibited a high specificity through Western blot. Tissue expression analysis showed that the expression of chicken SREBP1 was much higher in abdominal adipose tissue and heart, but lower in liver, muscle stomach, duodenum, spleen, kidney, and no signal was detected in breast and leg muscle. The SREBP1 expression characteristics between fat and lean broiler lines was also detected, and the result showed that SREBP1 expressed significantly higher in the abdominal adipose tissue of fat line than that in lean line (P<0.05). CONCLUSION: The antiserum against chicken SREBP1 prepared in this study had high titer and specificity. SREBP1 was highly expressed in abdominal adipose tissue of broilers. Compared to lean broiler line, SREBP1 was expressed higher in the fat line.