成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核...成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。展开更多
为了研究囊泡运输蛋白Mon1a(Mon1 secretory trafficking family member A)在细胞中的作用,该文利用类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)打靶技术构建了针对Mon1a基因的特定TALEN质粒对...为了研究囊泡运输蛋白Mon1a(Mon1 secretory trafficking family member A)在细胞中的作用,该文利用类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)打靶技术构建了针对Mon1a基因的特定TALEN质粒对,转染后筛选获得Mon1a基因敲除的HEK293T稳定细胞系。进一步从细胞增殖能力、迁移能力以及小鼠皮下成瘤能力等方面研究了Mon1a缺失对HEK293T细胞的影响。结果表明,Mon1a在细胞增殖、迁移和成瘤等过程中起到重要作用。展开更多
The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, method...The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout (KO) cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.展开更多
文摘成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。
文摘为了研究囊泡运输蛋白Mon1a(Mon1 secretory trafficking family member A)在细胞中的作用,该文利用类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALEN)打靶技术构建了针对Mon1a基因的特定TALEN质粒对,转染后筛选获得Mon1a基因敲除的HEK293T稳定细胞系。进一步从细胞增殖能力、迁移能力以及小鼠皮下成瘤能力等方面研究了Mon1a缺失对HEK293T细胞的影响。结果表明,Mon1a在细胞增殖、迁移和成瘤等过程中起到重要作用。
基金supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)
文摘The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout (KO) cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.