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.

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

“…Similarly, transfection of primary mouse embryonic fibroblasts (PMEFs) with siPtbp1/2 decreased the relative abundance of the intron 9-retained species 10-fold (Fig. 7D) and increased the (Khandelia et al 2011) were treated with 2 mg/mL Dox for 72 h, and the knockdown efficiencies were analyzed by RT-qPCR. Shown are residual mRNA expression levels normalized to the expression of the corresponding mRNAs in the presence of a firefly luciferase-specific shRNA control.…”

“…Expression of shRNAs was induced by treating corresponding CAD-A13 cell pools with 2 mg/mL Dox for 72 h as described (Khandelia et al 2011). To knock down Ptbp1 and Ptbp2 expression in PMEF cells, they were transfected with the corresponding siRNAs and RNAi MAX (Invitrogen) twice over a 36-h interval so that the cells were exposed to siRNA for 72 h in total.…”

“…Since transiently transfected Stx1b constructs failed to recapitulate the regulation, possibly as a result of titrating out a saturable nuclear factor (data not shown), we took advantage of the single-copy transgene knock-in technology based on high-efficiency and low-background recombination-mediated cassette exchange (HILO-RMCE) (Khandelia et al 2011). Using this approach, we generated a population of CAD cells expressing a ''minitransgene'' cassette in which a short intron 9-containing fragment of the Stx1b gene was placed under the control of a doxycycline (Dox)-inducible promoter element containing a recombinant constitutive intron (TREi) (Fig.…”

Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation

“…We used a site-specific recombinase technology to introduce ectopic tRNA Leu CAG into the genomic region. 46 Thus, the selection marker, blasticidin, of EV was replaced with a cassette containing hygromycin (selection marker) and tRNA Leu CAG , ruling out the variations caused by random insertions. The stable tRNA Leu CAG -expressing cell line showed faster growth and resistance to cell death than the stable EV-expressing cell line under normal as well as amino acid-depleted conditions (Fig.…”

“…46 The pEM584 plasmid (kindly provided by Dr. Eugene V. Makeyev) was first transfected into the NIH3T3 cell line, and colonies resistant to blasticidin S (2.5 μg/ml) were isolated, expanded and used as a control. The genomes of these colonies integrated with the RMCE cassette that contained the human EF-1α promoter and a blasticidin resistance gene (Bsd) “floxed” by the mutually incompatible Cre recombinase-specific sites, Lox2272 and LoxP.…”