Semi-quantitative analysis of transient single-cell gene expression in embryonic stem cells by femtoinjection.
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.
Saito
Mikako
M
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.
Matsuoka
Hideaki
H
D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D034622:RNA Interference / Q000502:physiology
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins