Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons.
RNA interference is an important tool for gene silencing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against doublecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons.
Yoon
Seung Yong
SY
Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Choi
Jung Eun
JE
Hwang
Onyou
O
Hong
Hea Nam
HN
Lee
Heuiran
H
Kim
Yoo Kyum
YK
Cho
Sung-Woo
SW
Kim
Hyun
H
Kim
DongHou
D
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D000089622:Doublecortin Domain Proteins; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D000089622:Doublecortin Domain Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins