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.

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.

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.

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.

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.

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.