biostudies-literatureUnknown108(31)Khandelia PSequence-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, NIHProceedings of the National Academy of Sciences of the United States of America12799-804https://www.ebi.ac.uk/biostudies/studies/S-EPMC3150880biostudies-literatureStreamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.PMC3150880Yap KKhandelia PMakeyev EVfalseStreamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.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, NIH2011-01-01T00:00:00Z2011 Aug2025-04-21T13:47:16.656Z2019-03-27T03:07:47ZS-EPMC31508802176839010.1073/pnas.1103532108