Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verif...Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction （PCR） using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 （DE3）. The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length （Genbank accession number： JX422071.1） with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology （99.6%） with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function.展开更多
[Objective] This study aimed to find out a method for low-cost and highly efficient sheep superovulation treatment and artificial insemination. [Method] The factors those probably influencing the results of convention...[Objective] This study aimed to find out a method for low-cost and highly efficient sheep superovulation treatment and artificial insemination. [Method] The factors those probably influencing the results of conventional superovulation and insemination, such as combination of FSH hormone and sponge suppository, estrus interval, number of insemination, and ram individuals were analyzed. [Result] The combination of sponge suppository and FSH produced in Beijing exhibited the poorest effect to superovulation, significantly worse than that of other combinations （P0.01）. The FSH produced in Ningbo, combined with sponge suppository or CIDR produced better effect to superovulation. The superovulation effect was better when the interval from the last FSH injection to estrus was 12 h, significantly better than that when the interval was 36 h （P0.01）; and there was no difference in the superovulation results when the interval was 0, 12 and 24 h. The pregnancy rate of two artificial inseminations was significantly higher than that of only one insemination （P0.01）. Rams themselves had significant influence on fertilization results. [Conclusion] The combination of domestic FSH and domestic sponge suppository cost much less and dose not reduce the superovulation results. Better fertilization result can be obtained if the ewes are inseminated twice with the sperm those gave high pregnancy rate.展开更多
[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead la...[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead lambs were collected and used to extract DNA for PCR with primers designed for N＋D1 fragment of follistatin gene.At the same time,we detected the CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector.The body tissues of dead lambs and muscle tissues of transgenic lambs in vivo were used to extract RNA for RT-PCR.[Result]The results showed that the DNA Extraction Kit was faster and more efficient than conventional method in extracting DNA and the DNA extracted with kit was easier to be amplified than that with conventional method.In order to avoid false positive caused by the interference of endogenous gene,the primers were designed for amplifying the combination of upstream of vector gene and downstream of target gene,increasing the specificity of detection.Tail tissue of newborn transgenic lambs could be used for detection and the detected results were reliable and accurate.The detection of CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector provided a data support for the biological safety of transgenic animals and verify the detected results of target gene of transgenic lambs.The transcription products of RNA extracted from three of the lambs were not detected.[Conclusion] The PCR method established in our research for detecting transgenic sheep was efficient,fast and accurate.It would provide experimental basis for further detection at protein level,lay a foundation for the establishment of multi-level and systematic detection method of transgenic sheep and provide a stable technology platform for safety monitoring of transgenic sheep.展开更多
基金Supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region(2012211B54)~~
文摘Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction （PCR） using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 （DE3）. The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length （Genbank accession number： JX422071.1） with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology （99.6%） with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function.
基金Supported by Science and Technology Project of Autonomous Regions(201111113)Science and Technology Projects of Xinjiang Autonomous Regions(201291147)Key Special Project for Breeding and Cultivation of GMO Varieties(2011ZX08008-003)~~
文摘[Objective] This study aimed to find out a method for low-cost and highly efficient sheep superovulation treatment and artificial insemination. [Method] The factors those probably influencing the results of conventional superovulation and insemination, such as combination of FSH hormone and sponge suppository, estrus interval, number of insemination, and ram individuals were analyzed. [Result] The combination of sponge suppository and FSH produced in Beijing exhibited the poorest effect to superovulation, significantly worse than that of other combinations （P0.01）. The FSH produced in Ningbo, combined with sponge suppository or CIDR produced better effect to superovulation. The superovulation effect was better when the interval from the last FSH injection to estrus was 12 h, significantly better than that when the interval was 36 h （P0.01）; and there was no difference in the superovulation results when the interval was 0, 12 and 24 h. The pregnancy rate of two artificial inseminations was significantly higher than that of only one insemination （P0.01）. Rams themselves had significant influence on fertilization results. [Conclusion] The combination of domestic FSH and domestic sponge suppository cost much less and dose not reduce the superovulation results. Better fertilization result can be obtained if the ewes are inseminated twice with the sperm those gave high pregnancy rate.
基金Supported by National Major Transgenic Project (2013ZX08008-003-04,2013ZX08010004-009)~~
文摘[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead lambs were collected and used to extract DNA for PCR with primers designed for N＋D1 fragment of follistatin gene.At the same time,we detected the CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector.The body tissues of dead lambs and muscle tissues of transgenic lambs in vivo were used to extract RNA for RT-PCR.[Result]The results showed that the DNA Extraction Kit was faster and more efficient than conventional method in extracting DNA and the DNA extracted with kit was easier to be amplified than that with conventional method.In order to avoid false positive caused by the interference of endogenous gene,the primers were designed for amplifying the combination of upstream of vector gene and downstream of target gene,increasing the specificity of detection.Tail tissue of newborn transgenic lambs could be used for detection and the detected results were reliable and accurate.The detection of CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector provided a data support for the biological safety of transgenic animals and verify the detected results of target gene of transgenic lambs.The transcription products of RNA extracted from three of the lambs were not detected.[Conclusion] The PCR method established in our research for detecting transgenic sheep was efficient,fast and accurate.It would provide experimental basis for further detection at protein level,lay a foundation for the establishment of multi-level and systematic detection method of transgenic sheep and provide a stable technology platform for safety monitoring of transgenic sheep.
