通过飞秒注射对胚胎干细胞中瞬时单细胞基因表达进行半定量分析。

Semi-quantitative analysis of transient single-cell gene expression in embryonic stem cells by femtoinjection.

作者信息

Saito Mikako, Matsuoka Hideaki

机构信息

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.

出版信息

Methods Mol Biol. 2010;650:155-70. doi: 10.1007/978-1-60761-769-3_13.

DOI:10.1007/978-1-60761-769-3_13

PMID:20686951

Abstract

Single-cell manipulation supporting robot (SMSR) has enabled femtoinjection, a high-throughput and semi-quantitative microinjection in the range of femtogram (fg) DNA and other molecules. An enhanced green fluorescent protein (EGFP) gene expression vector can be introduced directly into mouse embryonic stem cells at 100 cells/h with a 10% success rate. The intensity of EGFP fluorescence in single ES cells or single colonies of ES cells increases as the concentration of an EGFP gene expression vector in the injection capillary increases from 1 to 50 ng/muL. On the other hand, the knockdown of EGFP gene expression can be demonstrated by femtoinjection of siRNA against EGFP or an shRNA expression vector using an EGFP expressing ES cell line. Femotoinjection can provide a useful method for quantitative analysis of transient gene expression in single cells using RNAi.

摘要

单细胞操作辅助机器人（SMSR）实现了飞克注射，这是一种在飞克（fg）级DNA和其他分子范围内的高通量半定量显微注射。增强型绿色荧光蛋白（EGFP）基因表达载体可以以每小时100个细胞的速度直接导入小鼠胚胎干细胞，成功率为10%。随着注射毛细管中EGFP基因表达载体的浓度从1 ng/μL增加到50 ng/μL，单个ES细胞或ES细胞单克隆中的EGFP荧光强度会增加。另一方面，使用表达EGFP的ES细胞系，通过对EGFP的siRNA或shRNA表达载体进行飞克注射，可以证明EGFP基因表达的敲低。飞克注射可为使用RNAi对单细胞中瞬时基因表达进行定量分析提供一种有用的方法。