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. 2011 Aug 2;108(31):12799-804.
doi: 10.1073/pnas.1103532108. Epub 2011 Jul 18.

Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells

Piyush Khandelia  1 , Karen Yap, Eugene V Makeyev
Affiliations

Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells

Piyush Khandelia et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Establishing the HILO-RMCE acceptor cell lines. (A) Flowchart of a typical HILO-RMCE shRNA experiment. (B) Diagram of the HILO-RMCE reaction using the pRD1 donor plasmid. (C) The newly established acceptor lines were cotransfected in a 12-well (HEK293T-A2, HeLa-A12, HeLa-S3-A6, A549-A11, HT1080-A4, U2OS-A13, L929-A12, NIH 3T3-A7) or 6-well format (P19-A9, CAD-A13, N2a-A5) with a mixture containing 90% of pRD1 plasmid and 10% of a Cre-encoding plasmid (most cell lines, pCAGGS-Cre; NIH 3T3-A7, pCAGGS-nlCre) or the EGFP-encoding control plasmid pCIG. Following the puromycin selection, multiple colonies formed in the presence of Cre but not when Cre was substituted with EGFP.
Fig. 2.
Fig. 2.
Developing the HILO-RMCE technology. (A) Diagram of the HILO-RMCE reaction using the pRD-RIPE donor plasmid. (B) HeLa-A12 cells containing the RMCE acceptor locus were cotransfected in a 12-well format with the pRD-RIPE plasmid and the indicated amounts of the pCAGGS-Cre or pCAGGS-nlCre plasmids. Note that nlCre performed better than the wild-type Cre over a wide concentration range. (C) Genomic DNA was isolated from three parental cell lines (HEK293T, HeLa, and A549; lanes labeled “parent”), the corresponding HILO-RMCE acceptor clones (“A” followed by the clone number), and pooled clones obtained by the HILO-RMCE-mediated integration of the RIPE cassette (the “A+RIPE” lanes). The DNA samples were digested with NcoI and analyzed by Southern blotting to confirm the uniform rearrangement of the acceptor locus as a result of the RMCE reaction. The results are consistent with the expected increase in the length of the acceptor locus-specific NcoI fragment by 856 bp following the RIPE integration. (D) The precision of the RMCE reaction was further confirmed by analyzing the genomic DNA samples described in C by multiplex PCR using either the 5′ (EF, BR, and PR, see A) or the 3′ junction primer mixture (GF, BF, and WR; see A). The primers were designed so that the corresponding PCR product sizes were distinct for the original acceptor (5′-Bsd and Bsd-3′) and the RIPE-targeted loci (5′-Pur and EGFP-3′). GAPDH-specific primers detecting both the bona fide gene (GAPDH) and a pseudogene (ψGAPDH) were used as a control. (E) HILO-RMCE colonies produced by cotransfecting HEK293T-A2 and HeLa-A12 cells with pCAGGS-nlCre and either pRD1 or pRD-RIPE were pooled and incubated with 2 μg/mL Dox for 48 h or left untreated. The cellular EGFP expression was then examined by FACS. Note that nearly all cells express EGFP in the Dox-treated pRD-RIPE samples. (F) HEK293T-A2 cells carrying RIPE cassettes with shRNAs against either FLuc or LacZ were induced with Dox for 36 h or left untreated. The cells were then transfected with a mixture of plasmids encoding the FLuc and RLuc luciferases and the normalized FLuc activities were assayed as described in SI Materials and Methods. Data are averaged from six transfection experiments ± SD.
Fig. 3.
Fig. 3.
Silencing cell-encoded genes. (A and B) HEK293T-A2 cells containing four different RIPE-encoded shRNAs against human PTBP1 mRNA or the shFLuc shRNA were induced with Dox for 72 h and the efficiency of the PTBP1 knockdown analyzed by (A) RT-qPCR and (B) immunoblotting with PTBP1-specific antibodies. The RT-qPCR graph in A shows relative PTBP1 expression levels normalized to the shFLuc control. (C–F) The experiment in A and B was repeated in N2a-A5 cells using shRNAs against (C and D) mouse Ptbp1 or (E and F) Ago2 genes. Note that coexpressing the two most potent Ago2-specific shRNAs from a single RIPE cassette further improves the protein knockdown (lane “sh1+sh4” in F). (G and H) Optimization of the human TUT4/ZCCHC11 knockdown as a part of a larger-scale RNAi experiment where an shRNA library against the human TUT gene family was integrated into HEK293T-A2 cells using HILO-RMCE (see Fig. S6 for the rest of the results). (G) RT-qPCR analysis, in which the TUT4/ZCCHC11 mRNA expression levels in the Dox-treated samples were normalized to the corresponding Dox-negative controls. (H) The knockdown efficiencies were also studied by immunoblotting with an anti-TUT4 antibody. Data in A, C, E, and G are averaged from three amplifications experiments ± SD. In B, D, F, and H, a GAPDH-specific antibody was used to control lane loading.
Fig. 4.
Fig. 4.
Knocking down Ptbp1 in HILO-RMCE-generated populations modifies cellular alternative pre-mRNA splicing patterns. CAD-A13 cells containing RIPE-encoded shRNAs against mouse Ptbp1 (sh2 or sh4) or the shFLuc shRNA were induced with 2 μg/mL doxycycline and the time course of the Ptbp1 mRNA knockdown was followed for 108 h using RT-qPCR (A, Left). We also examined the time course of the Ptbp2 mRNA accumulation (A, Right), an expected outcome of the reduced Ptbp1 abundance (–30). (B) The Ptbp1 down-regulation and the Ptbp2 up-regulation were also confirmed by immunoblotting with corresponding antibodies. (C) RT-PCR analysis of the 72-h induced samples were carried out to examine the splicing patterns of three alternative cassette exons known to be repressed by the Ptbp1 protein: exon 10 of the Ptbp2 gene, exon N1 of the Src gene, and exon 5 of the Cltb gene. Note that the inclusion of these exons is stimulated in the Ptbp1-knockdown samples compared with the shFLuc control. i, exon-included splice form; s, exon-skipped splice form. Gapdh, an RT-PCR amplification control.
Fig. 5.
Fig. 5.
HILO-RMCE can be readily adapted for rapid engineering of transgenic cell pools. HEK293T-A2 cells were cotransfected with pCAGGS-nlCre and pRD1-based donor plasmid (pEM705) containing a CAG promoter-driven bicistronic cassette encoding dTomato (dTom) (37) and a nuclear localized EGFP proteins. Recombinant cells were selected with puromycin for 7 d, pooled and propagated for another 4 d. The fluorescent protein expression was then visualized by microscopy. Diagram of pEM705 is shown on the top of the panel.
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