In vivo electroporation has served as an effective tool for the study of developmental biology. Here we report tetracycline inducible gene knockdown by electroporation. Our system consists of genome integration of a cassette encoding long double strand RNA (dsRNA) of a gene of interest by electroporation, transcription of which is assured by RNA polymerase II, and induction of transcription of dsRNA by tetracyclin. Long dsRNA decapped by ribozyme in the cassette and without poly A tail is processed into siRNA within nuclei. We could successfully induce knockdown of En2 and Coactosin by Dox administration.
In vivo electroporation has been successfully applied in many model animals in the field of developmental biology as a tool of gene transfer in living embryos (Muramatsu et al. 1997; Funahashi et al. 1999; Nakamura 2009). Recently, revolutionary techniques have been added to gene transfer systems by electroporation. The first is transposon-mediated genome integration of a transgene (Sato et al. 2007), which enables long term and stable expression of a gene of interest. The other one is introduction of a Tet-on and Tet-off system to control transgene expression by tetracycline (Hilgers et al. 2005; Watanabe et al. 2007). Combination of transposon and Tet-on and Tet-off system has enabled long term control of transgene expressions.
When gene silencing by small interfering RNA (siRNA) became possible in higher vertebrates (Caplen et al. 2001; Elbashir et al. 2001; Harborth et al. 2001), we could successfully knockdown a gene of interest by electroporating short hairpin RNA (shRNA) expression vectors (Katahira & Nakamura 2003). But the expression of shRNA is dependent on RNA polymerase III, which assures ubiquitous expression so that spatial and temporal control of shRNA expression is difficult. Still, conditional knockdown by manipulating promoters for RNA polymerase III to express shRNA by plasmid and lentivirus vectors has been tried (Czauderna et al. 2003; Amar et al. 2006; Kappel et al. 2006; Desclaux et al. 2009). Conditional shRNA expression from the pol II promoter by adopting mir30-based shRNA expression system was also developed (Matsuda & Cepko 2007), but these systems had not been applied to knockdown endogenous embryonic gene expression. We have long desired to knockdown a gene of interest at the desired stage of development in a convenient way.
Successful knockdown of a target gene by siRNA that is processed from long double-strand RNA (dsRNA) transcribed by RNA polymerase II was developed (Shinagawa & Ishii 2003). The deletion of cap structure and poly(A) (pDECAP) vector contains the cytomegalovirus (CMV) promoter, a ribozyme cassette to cut off the m7G cap structure, and a MYC-associated zinc finger protein (MAZ) site for Pol II pausing (Yonaha & Proudfoot 1999, 2000). Since transcribed RNA lacks the cap and poly A tail, it stays in the nucleus, where long dsRNA is processed into siRNA by ribonuclease(s) such as Dicer.
In the present study, we tried to conditionally knockdown the target gene expression by siRNA that was processed from long dsRNA of which transcription is temporally controlled by tetracycline, taking advantage of Pol II dependent transcription. En2 and Coactosin were chosen as target genes. En2 is a transcription factor, and expressed in the metencephalon and mesencephalon (Patel et al. 1989). Coactosin is an actin binding protein, and is expressed in young neurons in the neural tube, in neural crest cells, and in axons of the oculomotor nerve (Hou et al. 2009).
Materials and methods
Construction of siRNA expression vector
For the construction of siRNA expression vector, a 426-nt chick Coactosin coding sequence, and 591-nt En2 coding sequence (125–715) separated by the 12-nt spacer (GGTGCGCATATG) from the reverse complementary sequence was inserted in the pDECAP vector (Shinagawa & Ishii 2003). A 12-nt non-palindromic spacer as a loop may facilitate the cloning and the hairpin formation. For construction of Coactosin dsRNA expression vecor, Coactosin was first inserted in pDECAP, then the DECAP cassette containing Ribozyme and MAZ sequence was blunted and inserted in the pT2K-BI-TRE-EGFP (Clonetech) with EcoRV (pT2K-BI-TRE-EGFP-DECAP-Coactosin) (Fig. 1). For construction of En2 dsRNA expression vector, En2 was inserted in pDECAP, then pDECAP was digested by BssHI, and inserted in MluI site of pT2K-BI-TRE-EGFP (pT2K-BI-TRE-EGFP-DECAP-En2). The DECAP cassette containing inverted repeat was amplified in Escherichia coli Sure 2 strain (Stratagene), or GT116 strain (Invivogen), which allows the accurate replication of DNA containing inverted repeats.
It was reported that dsRNA of longer than 400 nt were effective for gene silencing, but shorter than 100 nt had less effect in Drosophila (Hammond et al. 2000; Shinagawa & Ishii 2003). So, we used the whole open reading frame of Coactosin (426 nt), and a part of the open reading frame of En2 (591 nt).
In ovo electroporation for transfection of siRNA expression vector
Transfection was carried out by in ovo electroporation as described previously (Funahashi et al. 1999; Odani et al. 2008). Briefly, pT2K-BI-TRE-EGFP-DECAP-Coactosin (2.8 μg/μL) or pT2K-BI-TRE-EGFP-DECAP-En2 were mixed with pCAGGS-T2TP (2.4 μg/μL) and pT2K-CAGGS-rtTA-M2 (1.6 μg/μL), and were inserted into the lumen of brain vesicles or the trunk neural tube of stage 10 or 11 chick embryos, and a rectangular pulse of 25 V, 50 ms/s, that is, a pulse of 50 ms duration and 950 ms interval, was charged four times. Dox (200–300 μL of 0.2 mg/mL) was administered in the endodermal cavity (just beneath the embryo) at 12 h after electroporation.
For comparison, Pol III mediated knockdown of En2 was carried out by electroporating En2-150, in which the short hairpin of the 19-mer of oligonucelotide of the En2 open reading frame was inserted into the pSilencer1.0-U6 (Ambion) (Katahira & Nakamura 2003).
In some cases, green fluorescent protein (GFP) expression vector, pCAGGS-EGFP, was co-electroporated to show transfected cells.
In situ hybridization and Immunohistochemistry
Effects of siRNA were assessed by in situ hybridization and immunohistochemistry after fixation of embryos in 4% paraformaldehyde. Whole-mount in situ hybridization was performed as described by Bally-Cuif et al. (1995) or by Stern (1998). For immunohistochemistry, 4D9, anti-En2 monoclonal antibody (DSHB), and anti-Coactosin antibody that was raised in rabbits using bacterially expressed peptide, NH2-DHKELDEDYIKNELK-COOH, (Sawaday Technology), were used as primary antibodies. Specificity of the antibody was checked by Western blot; detection with anti-Coactosin and anti-GFP antibody on NIH3T3 cells that were transfected with pEGFP-Coactosin showed the same 44 kDa band. Secondary antibodies used were anti-mouse Alexa-594 (Invitrogen), and anti-mouse Alexa-488 (Invitrogen), anti-rabbit Alexa-594 (Invitrogen).
First, we checked whether dsRNA transcription from the Pol II promoter could be induced in vivo by Doxycycline (Dox), a tetracyclin derivative, then checked if it worked as siRNA with anti-En2 antibody (4D9). Since the transfected pT2K-BI-TRE-EGFP-DECAP-En2 vector assures both GFP expression and long double strand RNA, GFP expressing cells were regarded as those of interest. At 12 h after electroporation, when Dox was administered, no effects on En2 expression were discerned in all the embryos where pT2K-BI-TRE-EGFP-DECAP-En2 was electroporated, which was assessed by co-electroporated pCAGGS-EGFP vector (Fig. 2C,D; n = 4). At the same time, En2 knockdown by En2-150 had already occurred in all of the embryos electroporated (n = 3), which was driven by Pol III (Fig. 2A,B). After 24 h of Dox administration, En2 knockdown in the GFP expressing cells occurred in three out of four embryos electroporated (Fig. 2E,F). Without Dox administration, En2 knockdown did not occur in all embryos electroporated even at 36 h after electroporation (n = 3).
We then checked interference against Coactosin. GFP expression is detectable by 6 h after Dox administration as shown by Watanabe et al. (2007), that is, a relative amount of dsRNA is produced, although it is not clear if dsRNA is processed into siRNA. At that time, effects on Coactosin expression was hardly discernible in six embryos examined. At 12 h after Dox administration, five embryos out of 12 that were electroporated with the vector set, showed strong GFP fluorescence, and were fixed. In all five embryos, Coactosin expression was knocked down in GFP-expressing neural crest cells (1–4 in Fig. 2G,H). Coactosin was also knocked down in the neural tube (5, 6 and asterisk in Fig. 2G). Specificity of the siRNA was ascertained by the effect to Drebrin, a member of the actin-depolymerizing factor-homologous (ADF-H) family, where Coactosin belongs (Hou et al. 2009). Coactosin mostly consists of the ADF domain, and Coactosin and Drebrin share 50% DNA sequence homology of the ADF region. Drebrin was not affected by Coactosin dsRNA in all four embryos examined (Fig. 2I,J).
Next, we looked at the time course after Dox administration in cultured neural crest cells. First, embryos were electroporated in ovo in the trunk neural tube with pCAGGS-T2TP, pT2K-BI-TRE-EGFP-DECAP-Coactosin and pT2K-CAGGS-rtTA-M2, and reincubated for 12 h for the embryos to reach stage 14. Then embryos were taken out, and the neural tube of the transfected region was digested in 1.5 μg/mL Dispase (GIBCO), and cut into small pieces. The pieces of neural tube were plated onto cover slips coated with fibronectin (BD Bioscience) (10 μg/mL), and cultured. Neural crest cells migrate out from the neural tube (Newgreen & Thiery 1980). Dox was administered at a concentration of 1 μg/mL after 12 h of the onset of the culture.
Green fluorescent protein fluorescence could be detected 6 h after Dox administration, and fluorescing cells increased as time passed (Fig. 3A–D). The specimen was fixed after 28 h and processed for immunohistochemistry with anti-Coactosin antibody for evaluation of the interfering effect of dsRNA. As shown in Figure 3E–G, in almost all of the GFP expressing cells, that is, those Coactosin dsRNA producing cells, except for two cells in the field, Coactosin was knocked down.
Owing to in vivo electroporation we can now conveniently carry out gain- and loss-of-function experiments (Nakamura 2009). Recently, transposon mediated genome integration, and regulation of transgene by tetracycline were introduced in this system (Hilgers et al. 2005; Sato et al. 2007; Watanabe et al. 2007). These methods assure stable and long term expression of the transgene, and also control of transgene expression by tetracycline. Knockdown of a gene of interest is possible by electroporating shRNA expression vector (Katahira & Nakamura 2003; Nakamura et al. 2004). But shRNA is transcribed by the RNA polymerase III, and it is difficult to control its expression. It was shown that long dsRNAs that lack 5′-cap structure and polyA tail are not exported to the cytoplasm, and are processed into siRNA while they stay in the nucleus (Shinagawa & Ishii 2003). Since dsRNA expressed from the DECAP cassette is exported to the cytoplasm after having been cut into short dsRNA, it does not induce interferon reaction (Shinagawa & Ishii 2003). They showed that phosphorylation level of eIF2α, which is a substrate of the dsRNA-activated PKR protein kinase and indicates interferon reaction, was not enhanced in the 293T cells after pDECAP-Ski transfection. It was also shown that dsRNA from DECAP cassette does not induce nonspecific cell death (Shinagawa & Ishii 2003). We confirmed that pT2K-BI-TRE-EGFP-DECAP-Coactosin did not induce cell death by 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) staining (data not shown).
In the present study, we could successfully control knockdown of target genes; knockdown was achieved only after Dox administration (Fig. 2). Without Dox administration, En2 knockdown did not occur in the cells where pT2K-BI-TRE-EGFP-DECAP-En2 vector set that encodes En2 dsRNA is transfected. As we expected, long dsRNA that is decapped and without poly A tail may have been cut by ribonuclease(s) such as Dicer, and have been processed into short dsRNA in the nucleus, then processed short dsRNA may have moved to the cytosol to induce degradation of target mRNA (Shinagawa & Ishii 2003). By adopting this system, we have realized Tet-on of the siRNA system. In addition, since the cassette is integrated into the host genome, the system enables long term control of gene silencing in a very convenient way. Conditional knockdown by plasmid and lentivirus vectors driving shRNA expression has been tried (Czauderna et al. 2003; Amar et al. 2006; Kappel et al. 2006; Desclaux et al. 2009). Conditional shRNA expression by tetracycline inducible U6 or H1 promoters from the plasmid vector has been mainly tried in cell line, where transfection was achieved by two step; first Tet repressor-coding plasmid was transfected into the target cell line, then clones expressing high levels of TetR were selected and subsequently transfected with Tet-regulatable shRNA expression plasmid. Matsuda & Cepko (2007) developed the mir30-based shRNA expression system, of which expression is from the pol II promoter and is controlled by Cre-loxP system, where knockdown of transferred GFP was successful. But this system has not been applied for knockdown of the endogenous gene expression. These systems are worth trying for knockdown of the endogenous embryonic gene expression, since the short dsRNA is more convenient to handle than the long one. In the lentivirus vector system, entire Tet sets are in a single plasmid, so that the system is also worth trying to knockdown endogenous gene expression by electroporation.
Time course analysis of GFP revealed that GFP fluorescence became detectable at 6 h after Dox administration, and GFP expressing cells increased as time passed. Immunohistochemistry with anti-Coactosin and anti-GFP antibodies indicates that long dsRNA may have been well processed into siRNA and interfered with Coactosin expression. In almost all of the cells that express GFP, Coactosin expression was knocked down in culture. Control of knockdown by Dox administration is also effective in vivo.
In the present study, we have shown temporal control of knockdown of a gene of interest. Since the cassette is integrated into the genome by transposon, the method assures long term control of gene silencing in a convenient way. If we electroporated only Tet inducible vector, it assures short term conditional knockdown of a gene of interest. Spatial control of knockdown by using tissue specific promoter was already shown (Shinagawa & Ishii 2003). Thus, spatial and temporal control can be achieved conveniently by combining these methods, and the method presented here will be a strong tool for the study of developmental biology.
This work was supported by a Grant-in-Aid for scientific Research No. 17023003 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. cDNA of Drebrin and antibody against Drebrin were kindly provided by Professor Tomoaki Shirao.