Aquaculture has been believed to be a major Chinese contribution to the world. In recent 20 years, genome and other genetic technologies have promoted significant advances in basic studies on molecular basis and genet...Aquaculture has been believed to be a major Chinese contribution to the world. In recent 20 years, genome and other genetic technologies have promoted significant advances in basic studies on molecular basis and genetic improvement of aquaculture animals, and complete genomes of some main aquaculture animals have been sequenced or announced to be sequenced since the beginning of this century. Here, we review some significant breakthrough progress of aquaculture genetic improvement technologies including genome technologies, somatic cell nuclear transfer and stem cell technologies, outline the molecular basis of several economically important traits including reproduction, sex, growth, disease resistance, cold tolerance and hypoxia tolerance, and present a series of candidate trait-related genes. Finally, some application cases of genetic improvement are introduced in aquaculture animals, especially in China, and several development trends are highlighted in the near future.展开更多
Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat （CRISPR）/CRIS...Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated （Cas） system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts （PFFs） enabled inducing gene knock out （KO） in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination （HR）. With the aid of somatic cell nuclear transfer （SCNT） technology, three piglets with the biallelic lgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the lgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.展开更多
随着基因组学、基因组编辑技术的迅速发展以及显微注射技术、体细胞克隆技术的广泛应用,一套新型的育种策略和方法已经逐渐形成。这一套新型育种策略和方法可以称为分子编写育种(breeding by molecular writing,BMW)。该方法可以高效创...随着基因组学、基因组编辑技术的迅速发展以及显微注射技术、体细胞克隆技术的广泛应用,一套新型的育种策略和方法已经逐渐形成。这一套新型育种策略和方法可以称为分子编写育种(breeding by molecular writing,BMW)。该方法可以高效创制新的遗传标记并对其进行快速验证,也可以对基因组进行精确到分子水平的编写并定向培养新品种,不仅能打破生殖隔离,跨物种的引入新的性状,更可以对物种内个体间基因组进行精确到单个碱基的插入、删除和替换。如外源基因的精确整合,内源基因的精确删除、替换,SNP位点的复制、删除或替换等。该技术的优点是:可以在极大的降低非预期效应的同时,快速高效的将多种有益性状聚合到同一品种内。分子编写可进行以下四方面工作:(1)新型育种标记的创制及验证;(2)跨物种分子编写;(3)基因组中碱基序列的删除;(4)物种内分子编写。该育种技术可以不通过有性杂交,只引入一个或几个目标基因或SNP,快速获得目标性状突出的遗传稳定新种质,然后结合常规育种方法育成新品种。该方法将实现真正的个体和群体水平的基因(或分子)杂交育种,获得分子杂种优势,能够高效的解决长久以来困扰育种工作的诸多难题,大大提高育种效率,尤其在畜禽育种中具有重要应用前景,将会是未来育种的发展方向。文章详细论述了分子编写育种技术的基本概念、研究手段、研究内容、研究现状并展望了该技术的应用前景,为动物育种、畜禽繁殖等领域的研究及从业人员提供了参考。展开更多
High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the importa...High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.展开更多
为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TAL...为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。展开更多
基金supported by the National Key Basic Research Program of China (2010CB126301)the Special Fund for Agro-scientific Research in the Public Interest (200903046)the Autonomous Project of State Key Laboratory of Freshwater Ecology and Biotechnology (2011FBZ17)
文摘Aquaculture has been believed to be a major Chinese contribution to the world. In recent 20 years, genome and other genetic technologies have promoted significant advances in basic studies on molecular basis and genetic improvement of aquaculture animals, and complete genomes of some main aquaculture animals have been sequenced or announced to be sequenced since the beginning of this century. Here, we review some significant breakthrough progress of aquaculture genetic improvement technologies including genome technologies, somatic cell nuclear transfer and stem cell technologies, outline the molecular basis of several economically important traits including reproduction, sex, growth, disease resistance, cold tolerance and hypoxia tolerance, and present a series of candidate trait-related genes. Finally, some application cases of genetic improvement are introduced in aquaculture animals, especially in China, and several development trends are highlighted in the near future.
基金supported by a grant from the Jiangsu Key Laboratory of Xenotransplantation (BM2012116)
文摘Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated （Cas） system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts （PFFs） enabled inducing gene knock out （KO） in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination （HR）. With the aid of somatic cell nuclear transfer （SCNT） technology, three piglets with the biallelic lgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the lgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.
文摘随着基因组学、基因组编辑技术的迅速发展以及显微注射技术、体细胞克隆技术的广泛应用,一套新型的育种策略和方法已经逐渐形成。这一套新型育种策略和方法可以称为分子编写育种(breeding by molecular writing,BMW)。该方法可以高效创制新的遗传标记并对其进行快速验证,也可以对基因组进行精确到分子水平的编写并定向培养新品种,不仅能打破生殖隔离,跨物种的引入新的性状,更可以对物种内个体间基因组进行精确到单个碱基的插入、删除和替换。如外源基因的精确整合,内源基因的精确删除、替换,SNP位点的复制、删除或替换等。该技术的优点是:可以在极大的降低非预期效应的同时,快速高效的将多种有益性状聚合到同一品种内。分子编写可进行以下四方面工作:(1)新型育种标记的创制及验证;(2)跨物种分子编写;(3)基因组中碱基序列的删除;(4)物种内分子编写。该育种技术可以不通过有性杂交,只引入一个或几个目标基因或SNP,快速获得目标性状突出的遗传稳定新种质,然后结合常规育种方法育成新品种。该方法将实现真正的个体和群体水平的基因(或分子)杂交育种,获得分子杂种优势,能够高效的解决长久以来困扰育种工作的诸多难题,大大提高育种效率,尤其在畜禽育种中具有重要应用前景,将会是未来育种的发展方向。文章详细论述了分子编写育种技术的基本概念、研究手段、研究内容、研究现状并展望了该技术的应用前景,为动物育种、畜禽繁殖等领域的研究及从业人员提供了参考。
基金the National Basic Re-search Program of China (973 Program) (No. 2006CB504004 and 2006CB944004)the National Natural Science Foundation of China (No. 30430530)the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-YW-N-017).
文摘High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.
文摘为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。
