title: Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.
abstract: Sequence-specific gene silencing by short hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider biomedical use of this approach is hindered by the lack of straightforward methods for achieving uniform expression of shRNAs in mammalian cell cultures. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields virtually homogeneous cell pools containing doxycycline-inducible shRNA elements in a matter of days and with minimal efforts. To ensure immediate utility of this approach for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, HeLa-S3, and U2OS) and mouse (CAD, L929, N2a, NIH 3T3, and P19) cell lines to be compatible with the HILO-RMCE process. Because of its technical simplicity and cost efficiency, the technology will be advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that HILO-RMCE will facilitate a wider range of molecular and cell biology applications by allowing one to rapidly engineer cell populations expressing essentially any transgene of interest.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Piyush
authors.initials: P
authors.lastname: Khandelia
authors.forename: Karen
authors.initials: K
authors.lastname: Yap
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Makeyev
mesh_terms: D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; C450006:PTBP1 protein, human; D034741:RNA, Small Interfering; D038941:Polypyrimidine Tract-Binding Protein; D049452:Green Fluorescent Proteins; D008156:Luciferases