Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector.
Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.
Brown
Cheryl Y
CY
Women's and Children's Health Research Institute, Adelaide, South Australia 5006, Australia.
Sadlon
Timothy
T
Gargett
Tessa
T
Melville
Elizabeth
E
Zhang
Rui
R
Drabsch
Yvette
Y
Ling
Michael
M
Strathdee
Craig A
CA
Gonda
Thomas J
TJ
Barry
Simon C
SC
D000818:Animals; D004318:Doxycycline / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000235:genetics; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D018390:Gene Targeting; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D020598:Proto-Oncogene Proteins c-myb; D034741:RNA, Small Interfering; D004318:Doxycycline