Further evidence for heterozygote advantage of GJB2 deafness mutations:: a link with cell survival
Further evidence for heterozygote advantage of GJB2 deafness mutations:: a link with cell survival
复制标题
DOI：
10.1136/jmg.2003.017632
复制
发表时间：
2004-07-01
影响因子：
4
通讯作者：
Kelsell, DP
中科院分区：
文献类型：
--
作者：
Common, JEA;Di, WL;Kelsell, DP
方法将全长人野生型（WT）Cx 26和Cx 30独立地克隆到pEGFP-N3质粒（BD Biosciences Clontech）中。根据制造商的说明书，使用QuickChange SDM试剂盒（Stratagene）通过定点诱变（SDM）将疾病相关连接蛋白突变引入WT-Cx 26或WT-Cx 30中。所有阳性克隆均通过限制性内切酶分析鉴定，并进行DNA测序以检查是否发生错误的序列变化。根据制造商的说明书（Promega），使用transfast试剂将这些构建体转染到NEB 1角质形成细胞或NIH 3 T3成纤维细胞系中。48小时后，用培养基收获细胞，以收集所有活细胞和死细胞。然后将细胞储存在冰上直至荧光激活细胞扫描（FACS）分析。在分析前2分钟将碘化丙啶（PI）加入细胞中。对于每个样品，对10000个EGFP阳性细胞进行FACS分析，通过PI荧光指示该群体中的细胞死亡百分比（图1）。注意到NEB 1角质形成细胞亚群内源性表达Cx 26，以及其他表皮表达的Cx同种型，包括Cx 30、Cx 31和Cx 30。3.通过免疫细胞化学在NIH 3 T3成纤维细胞中检测到很少或没有内源性Cx 26，但是先前的实验证明了在表达Cx 31皮肤病相关构建体时这种细胞类型中的高细胞死亡计数。9
METHODSFull length human wildtype (WT) Cx26 and Cx30 were independently cloned into the pEGFP-N3 plasmid (BD Biosciences Clontech). The disease-associated connexin mutations were introduced into either WT-Cx26 or WT-Cx30 by site-directed mutagenesis (SDM) using the QuickChange SDM kit (Stratagene) according to the manufacturer’s instructions. All positive clones were identified by restriction enzyme analysis and DNA sequenced to check that no erroneous sequence changes had occurred. These constructs were transfected into either NEB1 keratinocyte or NIH 3T3 fibroblast cell lines using the transfast reagents according to the manufacturer’s instructions (Promega). After 48 h cells were harvested with the culture medium in order to collect all live and dead cells. Cells were then stored on ice until fluorescence-activated cell scanning (FACS) analysis. Propidium iodide (PI) was added to the cells 2 min prior to analysis. For each sample 10 000 EGFP positive cells were FACS analysed with the percentage of cell death indicated in this population by PI fluorescence (fig 1). It was noted that subpopulations of NEB1 keratinocytes express Cx26 endogenously, as well as other epidermally expressed Cx isoforms including Cx30, Cx31, and Cx30. 3. Little or no endogenous Cx26 was detected in the NIH 3T3 fibroblast cells by immunocytochemistry, but previous experiments demonstrated a high cell death count in this cell type upon expression of Cx31 skin disease-associated constructs. 9