[目的]通过CRISPR/Cas9技术构建KMT2D SET结构域双等位基因敲除的人胚胎干细胞HN4细胞系。[方法]通过CRISPR在线设计工具在SET结构域两侧各设计多条sgRNA,利用T7EI选出效率最高的sgRNA,构建到pX330载体中,特异性扩增同源臂,分别构建到...[目的]通过CRISPR/Cas9技术构建KMT2D SET结构域双等位基因敲除的人胚胎干细胞HN4细胞系。[方法]通过CRISPR在线设计工具在SET结构域两侧各设计多条sgRNA,利用T7EI选出效率最高的sgRNA,构建到pX330载体中,特异性扩增同源臂,分别构建到双抗性载体中作为Donor。将sgRNA与Donor共同电转至HN4细胞中,经过Puro和Hygro抗性筛选后,挑取单克隆,利用PCR,测序以及q-PCR鉴定等方法鉴定细胞的基因型。[结果]成功构建了靶向KMT2D SET Domain两端的sgRNA的表达质粒,并选择出切割效率最高的sgRNA。PCR鉴定结果显示,有8株细胞可同时扩增出Puro和Hygro两个目的片段,且无野生型条带。通过测序和序列比对确认KMT2D SET已被敲除,双等位基因分别被Puro和Hygro替换。q-PCR结果显示,mRNA层面上,SET Domain已经不表达,而其他位点则不受影响。[结论]成功构建pX330-sgRNA质粒,并验证其具有活性。成功构建了靶向KMT2D SET Domain的分别含有Puro和Hygro不同抗性的Donor。成功建立了稳定的KMT2D基因敲除的人胚胎干细胞系。展开更多
Kabuki syndrome(MIM 147920)is an autosomal dominant rare disease featured with multiple malformations and mental retardation.The main clinical manifestations of Kabuki syndrome are characteristic facial features,skele...Kabuki syndrome(MIM 147920)is an autosomal dominant rare disease featured with multiple malformations and mental retardation.The main clinical manifestations of Kabuki syndrome are characteristic facial features,skeletal abnormalities,dermatoglyphic abnormalities,postpartum growth retardation,mild to moderate mental retardation,as well as other structural and functional abnormalities that may involve multiple systems.The establishment of diagnosis needs to be combined with clinical phenotype and the discovery of pathogenic mutation.Compared with the abundant descriptions and records of genotype-phenotype of postpartum patients,few prenatal diagnosis cases of Kabuki syndrome had been reported,which partially result from lacking the knowledge of its phenotype in fetuses that might suggest the diagnosis.This report performed comprehensive prenatal examinations to identify a fetus's etiology with multiple structural anomalies characterized by ascites,thickening of local skin,and cardiac abnormalities.We ruled out intrauterine infection,thalassemia,and chromosome abnormality by corresponding tests.Finally,trio whole-exome sequencing revealed a de novo heterozygous variation c.15641g>A(p.r5214h)in exon 48 of the KMT2D gene was the fetus's genetic pathogeny causing Kabuki syndrome.This result suggests that Kabuki syndrome should be in the suspected etiology list for prenatal hydrops/ascites.Our study confirmed that prenatal whole-exome sequencing is an efficient tool for diagnosing fetal abnormalities,and a multidisciplinary team is necessary for providing pregnancy guidance to patients.展开更多
文摘[目的]通过CRISPR/Cas9技术构建KMT2D SET结构域双等位基因敲除的人胚胎干细胞HN4细胞系。[方法]通过CRISPR在线设计工具在SET结构域两侧各设计多条sgRNA,利用T7EI选出效率最高的sgRNA,构建到pX330载体中,特异性扩增同源臂,分别构建到双抗性载体中作为Donor。将sgRNA与Donor共同电转至HN4细胞中,经过Puro和Hygro抗性筛选后,挑取单克隆,利用PCR,测序以及q-PCR鉴定等方法鉴定细胞的基因型。[结果]成功构建了靶向KMT2D SET Domain两端的sgRNA的表达质粒,并选择出切割效率最高的sgRNA。PCR鉴定结果显示,有8株细胞可同时扩增出Puro和Hygro两个目的片段,且无野生型条带。通过测序和序列比对确认KMT2D SET已被敲除,双等位基因分别被Puro和Hygro替换。q-PCR结果显示,mRNA层面上,SET Domain已经不表达,而其他位点则不受影响。[结论]成功构建pX330-sgRNA质粒,并验证其具有活性。成功构建了靶向KMT2D SET Domain的分别含有Puro和Hygro不同抗性的Donor。成功建立了稳定的KMT2D基因敲除的人胚胎干细胞系。
基金supported by the Military Medicine Innovation Project(No.17JS001 and 18JS007),China.
文摘Kabuki syndrome(MIM 147920)is an autosomal dominant rare disease featured with multiple malformations and mental retardation.The main clinical manifestations of Kabuki syndrome are characteristic facial features,skeletal abnormalities,dermatoglyphic abnormalities,postpartum growth retardation,mild to moderate mental retardation,as well as other structural and functional abnormalities that may involve multiple systems.The establishment of diagnosis needs to be combined with clinical phenotype and the discovery of pathogenic mutation.Compared with the abundant descriptions and records of genotype-phenotype of postpartum patients,few prenatal diagnosis cases of Kabuki syndrome had been reported,which partially result from lacking the knowledge of its phenotype in fetuses that might suggest the diagnosis.This report performed comprehensive prenatal examinations to identify a fetus's etiology with multiple structural anomalies characterized by ascites,thickening of local skin,and cardiac abnormalities.We ruled out intrauterine infection,thalassemia,and chromosome abnormality by corresponding tests.Finally,trio whole-exome sequencing revealed a de novo heterozygous variation c.15641g>A(p.r5214h)in exon 48 of the KMT2D gene was the fetus's genetic pathogeny causing Kabuki syndrome.This result suggests that Kabuki syndrome should be in the suspected etiology list for prenatal hydrops/ascites.Our study confirmed that prenatal whole-exome sequencing is an efficient tool for diagnosing fetal abnormalities,and a multidisciplinary team is necessary for providing pregnancy guidance to patients.
