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Constitutive and conditional RNAi transgenesis in mice
Methods 53 (2011) 430–436
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Methods journal homepage: www.elsevier.com/locate/ymeth
Constitutive and conditional RNAi transgenesis in mice Aljoscha Kleinhammer a,1, Wolfgang Wurst b,c,d,1, Ralf Kühn a,c,⇑ a
Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany Max Planck-Institute of Psychiatry, Kraepelinstr. 2-12, 80804 Munich, Germany c Technical University Munich (Technische Universität München), Lehrstuhl für Entwicklungsgenetik, c/o Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. d Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE), Standort München, Schillerstr. 44, D-80336 München, Germany b
a r t i c l e
i n f o
Article history: Available online 22 December 2010 Keywords: RNAi Conditional shRNA Cre recombinase tetR/O Transgenic mice Rosa26 RMCE
a b s t r a c t Gene silencing by RNA interference (RNAi) has become a routine method for extracting function from the mammalian genome. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate RNAi both in cultured cells and mice and present therefore a fast alternative to conventional knockout approaches. We describe three strategies to control gene silencing in mice by shRNA expression that can be applied to any transcript of interest. The strategies include germline and inducible cell typespecific knockdowns, which depending on the molecular switch applied can be either permanent (Cre/ loxP) or reversible (tetO/tTA). For reliable expression the shRNAs of interest are knocked into a pre-engineered Rosa26 docking site by recombinase mediated cassette exchange (RMCE). ES cells expressing the shRNA of interest can then be used to generate shRNA transgenic mice. The high efficiency of RMCE in ES cells enables the fast production of knockdown mice for in vivo functional analysis. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Silencing of gene expression by RNA interference (RNAi) is used as standard tool for functional genomics in cultured mammalian cells. RNAi is a sequence-specific gene silencing process that occurs at the messenger RNA (mRNA) level. In invertebrate cells, long double stranded RNAs (dsRNA), which are processed into short interfering RNAs (siRNA) by the ribonuclease Dicer, induce efficient and specific gene silencing. In mammalian cells, long dsRNAs (>30 bp) elicit an interferon response resulting in the global inhibition of protein synthesis and non-specific mRNA degradation. However, short dsRNAs (<30 bp) trigger the specific knockdown of mRNAs in mammalian cells without interferon activation [1]. Such synthetic siRNAs can be introduced into cultured cells and induce a transient knockdown of target genes. Alternatively, RNA polymerase III driven expression vectors enable the permanent production of small dsRNAs in mammalian cells [2,3]. Such vector derived transcripts are designed to contain an antisense region, complementary to a selected mRNA sequence, and the corresponding mRNA sense region. These transcripts fold into a stem-loop structure and form short hairpin RNAs (shRNAs) that are processed by Dicer in a similar way as siRNAs. Expression vectors for shRNAs ⇑ Corresponding author. Fax: +49 0 89 3187 3099. E-mail addresses: [email protected] (A. Kleinhammer), [email protected] (W. Wurst), [email protected] (R. Kühn). 1 Fax: +49 0 89 3187 3099. 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.12.015
can be stably integrated into the genome and allow permanent gene silencing in cell lines and organisms. Like in cultured cells gene silencing can be transiently induced in mice by delivering siRNA to somatic tissues. Direct siRNA delivery is frequently used in experimental settings requiring only a short term knockdown of gene function [4], e.g. to study and the replication of Hepatitis C virus in the liver [5]. In clinical studies, local delivery of siRNAs have already proved of value as disease gene inactivating drugs [6–9]. Alternatively, permanent gene silencing can be achieved in mice or rats by integrating shRNA transgenes into the genome. The efficiency of gene silencing in transgenic mice can be as high as 90% and sometimes even higher. In several instances, corresponding knockdown and knockout phenotypes were compared and found to be identical or very similar [4,10,11]. As compared to somatic siRNA delivery, for which the uptake rate is critical, transgenic shRNA is expressed in all cells of the organism. shRNA transgene delivery to mice can be achieved by pronucleus injection, viral embryo infection or targeted insertion (knock-in) into the ES cell genome [11–14]. In this article we summarize our approach for a streamlined production of shRNA transgenic mice using targeted transgenesis in ES cells [10,13,15,16]. This approach involves the insertion of shRNA expression vectors into the transcriptionally active Rosa26 locus of ES cells by recombinase mediated cassette exchange (RMCE) followed by the generation of knockdown mice. The targeted insertion of shRNAs into the Rosa26 locus ensures reproducible and ubiquitous transgene expression. A single shRNA vector copy expressed from the Rosa26 locus is sufficient for
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pEx-H1tetO-CAG-tetR pRMCE-II pCAG-C31Int pRosa26-50 probe
constitutive, body-wide gene silencing [15]. However, tissuespecific knockdowns require conditional vectors equipped with a molecular switch. Most frequently this switch is provided by the Cre/loxP recombination system enabling both a spatial and temporal control of shRNA expression [13]. Alternatively the tetO/tTA system can be used to reversibly induce gene silencing in a temporally and spatially restricted manner [17].
Plasmid sequence information can be found at: http://www. rnai.ngfn.de/index_4.htm.
2. Materials
2.2. Cell lines
2.1. Plasmids
ES cell line IDG26.10-3 ES cell line IDG3.2 Feeder cells: G418 resistant murine embryonic fibroblasts Plasmids and cell lines are available from the authors
pbs-U6 pNeb-lox-stop-lox
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RMCE plasmid donor allele
attB neo
U6 shRNA
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M C R EE os S a c W 26. ell 10 cl T ES E on ce S c e lls el ls
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RMCE allele - 14 kb WT allele - 11 kb
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Fig. 1. Genomic integration of shRNA vectors by RMCE into the Rosa26.10 allele of ES cells. (A) An shRNA expression cassette and a neomycin resistance gene (neo), flanked by two attB recognition sites for C31 Integrase, are located on a donor plasmid that is transfected into Rosa26.10 ES cells. (B) The Rosa26 acceptor locus in IDG3.2 Rosa26.10 ES cells contains a hygromycin resistance coding region flanked by two attP recognition sites for C31 Integrase. PCR genotyping can be performed with the primer pair hyg1/ hyg2 resulting in a 550 bp PCR product. Using the Rosa-50 genomic hybridization probe and EcoRV (E) digestion, the acceptor allele is recognized as a 4.5 kb band. (C) Upon C31 Integrase mediated recombination of both pairs of attB and attP sites the hygromycin resistance gene becomes exchanged by recombinase mediated cassette exchange (RMCE) against the shRNA expression unit. ES cell clones habouring RMCE alleles can be selected by the neo resistance gene that is expressed from the pgk promoter within Rosa26. Recombined ES cell clones can be identified by PCR genotyping using the primer pair pgk/neo that amplifies a 280 bp fragment. Using the Rosa-50 genomic hybridization probe and EcoRV (E) digestion, the RMCE allele is recognized as a 14 kb band. (D) Southern blot analysis of genomic DNA derived from wildtype ES cells, Rosa26.10 acceptor ES cells and an ES cell clone habouring a RMCE (shRNA) allele. Using the Rosa-50 genomic hybridization probe (B, C) and EcoRV digestion the Rosa26 wildtype allele exhibits a 11 kb band, as compared to a 4.5 kb band derived from the Rosa26.10 acceptor allele and a 14 kb band derived from the Rosa26.10 RMCE (shRNA) allele.
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2.3. Primers Neo: (50 -GTT GTG CCC AGT CAT AGC CGA ATA G-30 ), Pgk (50 -CAC GCT TCA AAA GCG CAC GTC TG-30 ), Hyg-1 (50 -GAA GAA TCT CGT GCT TTC AGC TTC GAT G -30 ), Hyg-2 (50 -AAT GAC CGC TGT TAT GCG GCC ATT G -3’), Rosa-50 (50 -CGT GTT CGT GCA AGT TGA GT30 ), Rosa-30 (50 -ACT CCC GCC CAT CTT CTA G-30 ). 2.4. Reagents Doxycycline: Doxycycline hyclate (Sigma). Dissolve in water (1 mg/ml) and store in single-use aliquots at 20 °C. G418: Geneticin solution with an active concentration of 50 mg/ mL (Invitrogen). Store in single-use aliquots at 20 °C. Gelatine: 0.1% gelatine in water, autoclaved. 3. Description of method 3.1. Overview We describe here three RNAi approaches in transgenic mice to control gene knockdown by RNAi that can be applied to any
transcript of interest. These are: (i) constitutive and body-wide gene silencing, (ii) temporally and spatially controlled gene silencing based on Cre/loxP recombination and (iii) temporally controlled gene silencing based on the tetR/O system. In each case a shRNA expression construct is stably integrated into the genome of ES cells and subsequently transferred to the mouse germline. In these constructs the shRNA of interest is expressed from an RNA Polymerase III promoter. To obtain the double-stranded RNA configuration required for short hairpin RNA formation from single-stranded RNA we link a 19–22 bp ‘sense’ siRNA sequence to its complementary ‘antisense’ sequence via an 8 bp loop region and combine it with a poly T termination signal (Fig. 2A). Upon transcription the complementary RNA strands hybridize to form dsRNA that is required for Dicer recognition and gene silencing. The easiest way to obtain a clonable DNA fragment yielding a sense-loop-antisense configuration is by annealing of two complementary oligonucleotides shA and shB of about 60 bp. Oligonucleotides shA and shB are flanked by restriction enzyme recognition sequences that form restriction enzyme compatible overhangs after hybridization (Fig. 2A). For stable integration of an shRNA expression vector into the ES cell genome we use recombinase mediated cassette exchange (RMCE). Fig. 1 illustrates the C31Int/attB/attP mediated cassette
A loop
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Fig. 2. Generation of constitutive shRNA vectors for RMCE. (A) shRNA vectors are designed for U6 promoter driven expression in such a way that a 19–22 bp ‘sense’ siRNA sequence is linked to its complementary ‘antisense’ sequence by an 8 bp loop region and followed by a poly T termination signal. The corresponding single-stranded RNA transcript will form a double-stranded short hairpin (sh) RNA through self-hybridization. The shRNA sequence is obtained by designing two self-complementary oligonucleotides whose hybridization results in a cloneable fragment with restriction enzyme compatible overhangs. The green and red arrows must be replaced by the sense and antisense target gene sequence. The required G at position +1 is part of the vector so that the first base of oligonucleotide shA represents the second transcribed nucleotide (+2) of the shRNA. (B) Cloning scheme for the ligation of hybridized oligonucleotides shA + B into the vector pbs-U6 resulting in a vector for shRNA expression. AsiSi and Sfil digestion allows for the isolation of a 0.35 kb fragment that contains the shRNA expression cassette and is used in the next cloning step (C). (C) Cloning of constitutive shRNA vectors for RMCE in IDG26.10-3 ES cells. Plasmid pRMCE-II that is compatible for RMCE is opened with AsiSI and SfiI and ligated with the 0.35 kb fragment from pbs-U6-shRNA (B). The resulting pRMCE-U6-shRNA plasmid can be used for genomic integration of the shRNA construct in IDG26.10-3 ES cells.
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stable knockdown ES cell lines. Moreover, at least two transgenic mouse lines expressing two different shRNAs against the same target are required to rule out off-target effects. Mouse lines are deemed safe for further analysis if the knockdown phenotypes of the two shRNA mouse lines are similar [22,23]. Below we give step-by-step instructions for the production of constitutive and inducible shRNA knockdown ES cell lines and mice.
exchange at a pre-engineered Rosa26 docking site [13]. As mentioned above, Rosa26 is a ubiquitously transcribed locus in ES cells insuring stable and ubiquitous expression of any knocked-in transgene. Since shRNA donor cassettes include a promoterless neomycin resistance gene (neo) which is activated only after correct cassette exchange at the Rosa26 acceptor locus (Fig. 1C), RMCE positive cell lines can be conveniently selected in G418. A more detailed description of the C31Int/attP/attB site-specific recombination system can be found Brown et al. and Monetti et al. (this issue, pages 372 and 380, respectively). After stable integration, vectors expressing shRNAs constitutively will directly induce gene silencing. A more detailed description of the induction mechanisms can be found elsewhere [18,19]. For the conversion of constitutive shRNA vectors into Cre-inducible vectors, a loxP-stop cassette which disrupts its function is inserted into a unique HindIII restriction site engineered into the shRNA loop region (Fig. 3). Given the diversity of the already existing Cre and Cre-ER transgenic driver lines (see Smedley et al., this issue, page 411, [20]) the Cre inducible shRNA transgenes enable detailed gene analysis in the context of an entire organism. Unlike the Cre/ loxP system, the tet repressor/tet operator (tetR/O) switch is not dependent on enzyme-based DNA rearrangement but the small synthetic compound doxycycline (dox). In the absence of dox the tetracycline repressor (tetR), expressed from a constitutive promoter, binds to the tet operator (tetO) of a modified H1 promoter and blocks shRNA transcription (Fig. 4B). Exposure to dox dissociates the tetR from tetO which induces shRNA expression (Fig. 4B) [19]. Thus, gene silencing is induced by dox addition [17,21]. By following the protocols described below, constitutive and conditional knockdown ES cell lines can be obtained in 2 months and the corresponding knockdown mouse lines in additional 4 months. As a rule, the gene silencing performances of five predicted siRNAs per target need to be evaluated in ES cells before creating
3.2. Constitutive knockdown 3.2.1. Cloning of constitutive shRNA vectors 1. Select five siRNA target sequences for your gene of interest with siRNA prediction tools. For the constitutive and the Cre-inducible knockdown vectors the U6 gene promoter is used. Make sure to use Guanine as the first nucleotide of the siRNA sequence because it is required for efficient transcription from the U6 promoter. Free siRNA prediction programs are available at the web pages of Ambion, Invitrogen or Qiagen and the University of Tokyo (http://genomics.jp/sidirect/index.php? type=fc), University of Vienna (http://rna.tbi.univie.ac.at/cgibin/RNAxs) and the Whitehead Institute (http://jura.wi.mit.edu/bioc/siRNAext/home.php). 2. Design a pair of complementary oligonucleotides shA and shB for each siRNA target sequence and follow the design shown in Fig. 2A. The purchase of expensive phosphorylated oligonucleotides is unnecessary as the vector ends remain phosphorylated after the following ligation reaction. 3. Anneal complementary oligonucleotides shA/B (1 lg/ll) by diluting 1 ll shA and 1 ll shB in 98 ll TE and incubating at 100 °C for 5 min. Allow the annealing reaction to cool down slowly to room temperature within 20 min.
A AsiSI
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conditional shRNA Vector (loxP)
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pRMCE-U6lox/lox-shRNA
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conditional shRNA Vector (loxP) for RMCE
Fig. 3. Generation of conditional Cre/loxP shRNA vectors for RMCE. (A) Constitutive shRNA vectors (pbs-U6-shRNA, Fig. 2) can be turned into Cre-incucible conditional shRNA vectors by introduction of a removable loxP-flanked stop cassette (863 bp MlyI fragment from pNEB-lox-stop-lox) into the unique HindIII restriction site within the loop region of the shRNA construct. The 1.2 kb conditional shRNA expression cassette can be isolated from pbs-U6-lox/lox-shRNA after digestion with AsiSI and SfiI. (B) Cloning of Cre-incucible conditional shRNA vectors for RMCE in IDG26.10-3 ES cells. Plasmid pRMCE-II that is compatible for RMCE is opened with AsiSI and SfiI and ligated with the 1.2 fragment from pbs-U6-lox/lox-shRNA (A). The resulting pRMCE-U6-lox/lox-shRNA plasmid can be used for genomic integration of the shRNA construct into IDG26.10-3 ES cells.
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A loop
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Dox-inducible shRNA vector for RMCE
shRNA
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Fig. 4. Generation of conditional tetR/O shRNA vectors for RMCE. (A) Two self-complementary oligonucleotides shA/B including the shRNA sequence are designed and hybridized resulting in a cloneable DNA fragment with an BsaBI compatible 50 end as this enzyme produces blunt ends. Additionally, the fragment is AscI compatible due to its overhang at the 30 end. Green and red arrows are meant to be replaced by target gene sense and antisense sequences respectively. The loop region contains a unique HindIII site and ‘‘TTTTTT’’ serves as Pol III termination signal. (B) Cloning instructions for the generation of a conditional tetR/O shRNA vector by ligation of the annealed oligonucleotides shA/B into the vector pEx-H1-tetO-CAG-tetR opened with BsaBI and AscI. The generated vector pEx-H1-tetO-shRNA-CAG-tetR provides all necessary elements of the tetR/O system. As the vector contains in addition attB sites and a promoterless neomycin resistance gene it can be used directly for RMCE. In the absence of dox the tetracycline repressor (tetR), expressed from the ubiquitous active CAG promoter, binds to the tet operator (tetO) of the H1-tetO promoter and blocks shRNA transcription. Exposure to dox dissociates tetR from tetO and thereby activates the H1 promoter resulting in the production of shRNA.
4. Digest the pbs-U6 plasmid DNA with BseRI and BamHI. Ligate 2 ll hybridization reaction (step 3) with 100 ng opened pbsU6 in a 10 ll standard ligation reaction at 16 °C overnight (Fig. 2). Redigest the ligation with BamHI to eliminate selfligated empty vector. 5. Transform the ligation reaction into Escherichia coli cells (we use DH5a), and digest miniprep DNA with PstI and HindIII. Correctly ligated clones show two bands of 310 bp and 2.9 kb. Plasmids without insert show only the 2.9 kb vector band. M13 forward and M13 reverse primers can be used for sequence confirmation. 6. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) with one confirmed clone for each of the 5 predicted target sequences and use these plasmids in the experiments. 3.2.2. Pretesting of knockdown efficiency by transient transfection into ES cells 1. For pretesting the shRNA knockdown efficiency in vitro electroporate the five plasmids individually into IDG3.2 ES cells grown on gelatine coated plates. For transfection use 40 lg circular plasmid DNA to electroporate 4 106 ES cells in 0.8 ml PBS with a 0.4 cm BioRad cuvette (we use the BioRad Genepulser Xcell set at 330 V and 3 ms time constant). Plate transfected cells into a 10 cm gelatine coated culture dish and refresh medium at the next day. 2. Harvest cells two days after electroporation and determine the expression level of your target gene by Western blotting or real time PCR. 3. Select the two best performing shRNA vectors yielding at least a 70% knockdown of the target protein/mRNA and use these for creating stable ES cell lines.
3.2.3. Stable integration of shRNA vectors into ES cells by RMCE 1. The U6-shRNA expression cassette is ligated into the pRMCE-II vector that provides elements (neomycin resistance, attB sites) required for RMCE into Rosa26. Digest the pbs-U6-shRNA plasmid with AsiSI and SfiI and the pRMCE-II vector with AsiSI and SfiI and dephosphorylate the ends (Fig. 2C). Ligate the 350 bp AsiSI-SfiI U6-shRNA fragment derived from pbsU6-shRNA into the AsisSI/SfiI pRMCE-II vector (4.3 kb). 2. Transform into E. coli cells, prepare minipreps and identify correct clones by digesting with SalI and HindIII. Correctly ligated clones show two bands of 1.6 kb and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. M13 forward, M13 reverse and bpA-for primers can be used for sequence confirmation. 3. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) and use these two generated plasmids for RMCE in ES cells. 4. Co-electroporate 25 lg (circular) DNA of each plasmid from step 3. together with 25 lg (circular) DNA of the C31 Integrase expression vector pCAG-C3Int into IDG26.10-3 ES cells grown on feeder layers. For electroporation of 50 lg plasmid DNA use 4 106 ES cells in 0.8 ml PBS with a 0.4 cm BioRad cuvette (we use the BioRad Genepulser Xcell set at 300 V and 2 ms time constant). Plate transfected cells into two 10 cm culture dishes containing embryonic fibroblasts as feeder layer. 5. On day 2 after transfection add G418 (140 lg/ml) to the culture medium. Keep in selection for 8 days by changing medium daily. 6. On day 9: Pick 12 (or more) colonies per RMCE experiment. Expand cells on feeders for freezing and on gelatine for DNA isolation.
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7. Extract genomic DNA by standard procedures [24] and identify positive clones by PCR or Southern blotting as described below. 3.2.4. Identification of RMCE positive ES cell clones 1. For PCR identification of RMCE positive ES cell clones use the neo and pgk primers listed in Section 2.3 (annealing temperature: 65 °C; correct product size 280 bp). Note that feeder cells containing a pgk-neo resistance gene will yield an additional amplification product of 160 bp. Incorrectly recombined clones will still contain the hygro gene which can be checked by using the Hyg-1 and Hyg-2 primers listed in Section 2.3 (annealing temperature 65 °C, product size 550 bp). Note that using feeders harbouring an additional hygro resistance gene precludes this screening procedure. 2. Southern blot verification of correct clones is mandatory for exclusion of partially recombined alleles or chromosomal rearrangements. To distinguish the wildtype and RMCE Rosa26 alleles cleave the genomic DNA with EcoRV and use the 450 bp EcoRI fragment from plasmid pRosa-50 probe as hybridization probe. The wildtype Rosa26 locus is identified by a band of 11.5 kb (Fig. 1D). The parental IDG26.10-3 ES cells show an additional band of 4.5 kb derived from the modified Rosa26 allele before RMCE (Fig. 1D). Positive RMCE clones show an additional band of 14 kb due to the integration of the U6-shRNA and neo gene cassettes (Fig. 1D). Usually 40% of the neomycin resistant clones underwent complete RMCE. 3. Evaluate gene silencing efficiency of confirmed RMCE clones by subjecting aliquots to Western Blot and/or RT PCR analysis as described in Section 3.2.2. 4. Use confirmed clones for generating mouse lines by blastocyst injection or tetraploid embryo aggregation as described in [25]. 3.3. Cre-inducible conditional knockdown 3.3.1. Cloning of Cre/loxP conditional shRNA vectors 1. Open the vector pbsU6-shRNA (see Section 3.2.1) with HindIII, fill in ends and dephosphorylate the 50 -overhanging ends using standard procedures [24] (vector fragment size is 3.3 kb). 2. Prepare the 863 bp lox-STOP-lox cassette from the plasmid pNEB-lox-stop-lox plasmid by digesting with MlyI. 3. To generate plasmid pbs-U6-lox/lox-shRNA, ligate the fragment containing the lox-STOP-lox cassette from step 2 into the pbsU6-shRNA plasmid from step 1 and eliminate religated vector background by digesting with HindIII. 4. Transform the ligation product into E. coli cells and and screen minipreps for inserts by digesting with EcoRI and SacII. Correctly ligated clones show an 850 bp band for the insert and a 3.4 kb vector band. Inversely oriented inserts produce 150 bp and 4 kb bands. Clones without insert show only the vector band. Use M13 forward and M13 reverse primers for sequence confirmation. 5. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) for one confirmed clone. 6. Test the functionality of the lox-STOP-lox cassette as described below. 3.3.2. In vitro lox-STOP-lox cassette excision and testing shRNA knockdown efficiency in ES cells 1. Delete the lox-stop-lox cassette in vitro by incubating 1 lg conditional shRNA plasmid DNA with 4 Units of Cre recombinase (New England Biolabs) in a total volume of 50 ll 1x Cre buffer at 37 °C for 30 min. Inactivate the recombinase for 10 min at 70 °C.
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2. Eliminate non-recombined plasmids by EcoRI digestion. 3. Transform the reaction into E. coli cells and confirm recombination by digesting miniprep DNA with XbaI. Cre recombined plasmids that lost the stop cassette produce two bands of 460 bp and 2.9 kb. Nonrecombined plasmids produce bands of 1.3 kb and 2.9 kb. After sequence confirmation using the M13 forward and reverse primers prepare a Maxiprep (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) from one confirmed clone. 4. Pretest the activated shRNA plasmids by transiently transfecting the Maxiprep DNA obtained in step 3 into ES cells as described in Section 3.2.2. 5. Choose these constructs in the conditional state for ligation into RMCE vectors and subsequent stable transfection into ES cells by recombinase mediated cassette exchange. The knockdown efficiency of stably integrated vectors should be tested in ES cells as described for constitutive vectors in Section 3.2.4, step 3 with the additional necessity of activating the integrated vectors by Cre before testing them. 3.3.3. Stable integration of Cre/loxP conditional shRNA vectors into ES cells by RMCE For construction of Cre-inducible shRNA vectors compatible for RMCE follow the protocol for the construction of constitutive shRNA RMCE vectors (Section 3.2.3). Here we point out only those steps that differ to the protocol in Section 3.2.3, step 1: Ligate the U6-shRNA-lox-stop-lox fragment (1.2 kb) into the opened pRMCEII vector (4.3 kb). Section 3.2.3, step 2: Identify correct clones by plasmid digestion with SalI and EcoRI. Correctly ligated clones show two bands of 1.7 kb and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. Section 3.2.4, step 2: The wildtype Rosa26 locus can be identified by a band of 11.5 kb (Fig. 1D). The parental IDG26.10-3 ES cells show an additional band of 4.5 kb derived from the modified Rosa26 allele before RMCE (Fig. 1D). Positive RMCE clones show an additional band of 15 kb due to the integration of the U6-shRNA-lox-stop-lox and neo gene cassettes. Section 3.2.4: step 4, Use only confirmed ES cell clones for generating mouse lines by blastocyst injection or tetraploid embryo aggregation. Resulting mice need to be mated to Cre deleter strains for knockdown activation. 3.4. Doxycycline inducible knockdown This strategy employs vector pEx-H1tetO-CAG-tetR consisting of a tetO modified H1 promoter driving shRNA expression, a codon optimized tetR gene controlled by a constitutive CAG promoter and a promoterless neo resistance coding region. These elements are flanked by attB sites to enable RMCE (Fig. 4B). 3.4.1. Cloning of conditional tetR/O shRNA vectors 1. Select five siRNA target sequences for your gene of interest as described in Section 3.2.1. siRNA sequences may start with any nucleotide since in this approach the H1 and not the U6 promoter is used. 2. Design and anneal shA and shB oligonucleotides for each selected mRNA target sequence as described in Section 3.2.1, steps 1–3 except that BsaBI and AscI compatible ends are added (Fig. 4A). 3. Digest the plasmid pEx-H1tetO-CAG-tetR with BsaBI and AscI (Fig. 4B) and ligate the double stranded shA/B DNA to the vector. Redigest the ligation with BsaBI and AscI to remove the self-ligated empty vector. 4. Transform the ligation product into E. coli cells (we use DH5a) and digest miniprep DNA with PstI and HindIII. Correctly ligated clones should yield 4 bands of 0.2 kb, 1.0 kb, 2.8 kb and 3.3 kbs.
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Empty plasmids should yield only 3 bands of 0.2 kb, 1.0 kb and 6.1 kbs. M13 forward and reverse primers can be used for sequence confirmation as described in Section 3.2.4, step 3. 5. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) with one confirmed clone for each of the five predicted target sequences. 3.4.2. Pretesting of tetR/O shRNA vectors by transient transfection into ES cells Follow the steps described in Section 3.2.2 using 1 lg/ml dox in the culture medium to induce the knockdown. Select the two best performing shRNA vectors (>70% KD activity of the target) for RMCE. 3.4.3. Stable integration of tetR/O shRNA vectors into ES cells by RMCE 1. The pEx-H1tetO-shRNA-CAG-tetR plasmids generated in Section 3.4.1, step 6 can be directly used for RMCE in IDG26.10-3 ES cells. For RMCE follow steps 4–7 of Section 3.2.3. Identify RMCE positive subclones by PCR and Southern blot as described in Section 3.2.4. The wildtype Rosa26 locus can be identified by a band of 11.5 kb (Fig. 1D). The parental IDG26.10-3 ES cells show an additional band of 4.5 kb derived from the modified Rosa26 allele before RMCE (Fig. 1D). Positive RMCE clones exhibit a 17.7 kb band corresponding to the cassette exchange allele in addition to the 11.5 kb wild type allele. 2. Evaluate gene silencing efficiency as described in Section 3.2.4, step 3 using western blotting and/or or RT-PCR. For knockdown induction add 1 lg/ml doxycycline to the culture medium and set up and perform two negative controls in parallel. These are: (i) tetR/O shRNA positive cells kept in dox-free medium and IDG26.10-3 parental cells exposed to 1 lg/ml dox. The first control serves for estimating the extent of dox induction whereas the second reflects vector leakiness. 3. In mice knockdown activation is achieved by supplying the drinking water with 2 mg/ml dox and 10% sucrose. Light protection of dox containing water bottles is required; the water should be replaced every other day to maintain constant blood levels of dox. 4. Concluding remarks – how to use KD ES cell lines and mice for extracting function from the mammalian genome The shRNA vector approach described in this article enables body-wide and cell type-specific gene silencing in most tissues of adult mice. Particulary in the brain mRNA and protein levels can be reduced up to 90% with this technology [10,13,15,16]. However, the major factor for successful gene silencing in vivo is the preselection of highly effective shRNA sequences by in vitro siRNA screening. Compared to the generation of knockout mice by gene targeting the shRNA approach is technically less demanding and does not require homozygosity for the functional analysis of recessive genes. The knock-in insertion of shRNA vectors into a Rosa26 universal docking site enables the production of transgenic mice with reproducible shRNA expression patterns. Moreover, the generation of knockdown mice is relatively fast – a first group of adult mutants can be obtained within a period as short as 11 months [17,26,27]. The development of genetic mouse models that mimic human disease conditions to elucidate pathogenic mechanisms is the most direct application of in vivo RNAi [10,28]. For many complex diseases an increasing number of risk alleles and modifiers are being discovered by genome wide association studies and more recently also by the analysis of copy number variation [29]. Accordingly, the targeted modification of several genes within the same organism is highly desirable. Standard knockout technology can not be easily scaled up to more than two genes. Since gene silencing is indepen-
dent of a specific genomic target sequence, RNAi technology provides the means for simultaneously manipulating a significantly larger number of genes. For example, a single Rosa26 shRNA allele conferring a single gene knockdown can be easily combined with a second Rosa26 knockdown allele by simple breeding. If each of these alleles would be equipped with shRNAs directed against two different targets simple breeding could achieve knockdown of up to four target genes in the same organism by using only two transgenic loci that can be further freely combined with other knockdown units. Further transgenic RNAi applications include the validation of proteins as drug targets to predict the therapeutic potential of loss-of-function phenotypes [30–32]. Finally, modified siRNAs can be directly used as drugs and have proved successful in several clinical trials [6], most notably in the treatment of macular degeneration where the knockdown of vascular endothelial growth factor (VEGF) gene by local application of modified siRNAs significantly ameliorated the disease [33]. Acknowledgments This work has been funded by the Volkswagen Foundation and the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (FKZ: 01GR0404 and 01GS0858). We thank H. von Melchner for critical reading of the manuscript. References [1] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Nature 411 (2001) 494–498. [2] T.R. Brummelkamp, R. Bernards, R. Agami, Science 296 (2002) 550–553. [3] P.J. Paddison, A.A. Caudy, E. Bernstein, G.J. Hannon, D.S. Conklin, Genes Dev. 16 (2002) 948–958. [4] R. Kuhn, S. Streif, W. Wurst, Handb. Exp. Pharmacol. (2007) 149–176. [5] Q. Pan, H.W. Tilanus, H.L. Janssen, L.J. van der Laan, Expert Opin. Biol. Ther. 9 (2009) 713–724. [6] A.K. Vaishnaw, J. Gollob, C. Gamba-Vitalo, et al., A status report on RNAi therapeutics, Silence 1 (2010) 14. [7] D.M. Dykxhoorn, D. Chowdhury, J. Lieberman, Adv. Exp. Med. Biol. 615 (2008) 299–329. [8] D.M. Dykxhoorn, J. Lieberman, Cell 126 (2006) 231–235. [9] K. Tiemann, J.J. Rossi, EMBO Mol. Med. 1 (2009) 142–151. [10] S. Delic, S. Streif, J.M. Deussing, et al., Genes Brain Behav. 7 (2008) 821–830. [11] T. Kunath, G. Gish, H. Lickert, N. Jones, T. Pawson, J. Rossant, Nat. Biotechnol. 21 (2003) 559–561. [12] H. Hasuwa, K. Kaseda, T. Einarsdottir, M. Okabe, FEBS Lett. 532 (2002) 227–230. [13] C. Hitz, W. Wurst, R. Kuhn, Nucleic Acids Res. 35 (2007) e90. [14] D.A. Rubinson, C.P. Dillon, A.V. Kwiatkowski, et al., Nat. Genet. 33 (2003) 401– 406. [15] J. Seibler, B. Kuter-Luks, H. Kern, et al., Nucleic Acids Res. 33 (2005) e67. [16] P. Steuber-Buchberger, W. Wurst, R. Kuhn, Genesis 46 (2008) 144–151. [17] J. Seibler, A. Kleinridders, B. Kuter-Luks, S. Niehaves, J.C. Bruning, F. Schwenk, Nucleic Acids Res. 35 (2007) e54. [18] M. Lewandoski, Nat. Rev. Genet. 2 (2001) 743–755. [19] M. Wiznerowicz, J. Szulc, D. Trono, Nat. Methods 3 (2006) 682–688. [20] A. Nagy, L. Mar, G. Watts, Methods Mol. Biol. 530 (2009) 365–378. [21] M. van de Wetering, I. Oving, V. Muncan, et al., EMBO Rep. 4 (2003) 609–615. [22] E.M. Anderson, A. Birmingham, S. Baskerville, et al., RNA 14 (2008) 853–861. [23] A. Birmingham, E.M. Anderson, A. Reynolds, et al., Nat. Methods 3 (2006) 199– 204. [24] J. Sambrook, P. Macallum, D. Russell, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbour Press, Cold Spring Harbour, 2001. [25] S.W. Reid, L. Tessarollo, Methods Mol. Biol. 530 (2009) 269–285. [26] M.J. Herold, J. van den Brandt, J. Seibler, H.M. Reichardt, Proc. Natl. Acad. Sci. USA 105 (2008) 18507–18512. [27] K. Kotnik, E. Popova, M. Todiras, et al., PLoS ONE 4 (2009) e5124. [28] H. Zhou, B.H. Falkenburger, J.B. Schulz, K. Tieu, Z. Xu, X.G. Xia, Int. J. Biol. Sci. 3 (2007) 242–250. [29] L. Fugger, M.A. Friese, J.I. Bell, Nat. Rev. Immunol. 9 (2009) 408–417. [30] T. Christoph, G. Bahrenberg, J. De Vry, et al., Mol. Cell. Neurosci. 37 (2008) 579– 589. [31] R. Sacca, S.J. Engle, W. Qin, J.L. Stock, J.D. McNeish, Methods Mol. Biol. 602 (2010) 37–54. [32] R. Yang, G. Castriota, Y. Chen et al., Int. J. Obes. (Lond)., 2010, doi:10.1038/ ijo.2010.128. [33] L. Singerman, Retina 29 (2009) S49–S50.
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Constitutive and conditional RNAi transgenesis in mice Aljoscha Kleinhammer a,1, Wolfgang Wurst b,c,d,1, Ralf Kühn a,c,⇑ a
Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany Max Planck-Institute of Psychiatry, Kraepelinstr. 2-12, 80804 Munich, Germany c Technical University Munich (Technische Universität München), Lehrstuhl für Entwicklungsgenetik, c/o Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. d Deutsches Zentrum für Neurodegenerative Erkrankungen e. V. (DZNE), Standort München, Schillerstr. 44, D-80336 München, Germany b
a r t i c l e
i n f o
Article history: Available online 22 December 2010 Keywords: RNAi Conditional shRNA Cre recombinase tetR/O Transgenic mice Rosa26 RMCE
a b s t r a c t Gene silencing by RNA interference (RNAi) has become a routine method for extracting function from the mammalian genome. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate RNAi both in cultured cells and mice and present therefore a fast alternative to conventional knockout approaches. We describe three strategies to control gene silencing in mice by shRNA expression that can be applied to any transcript of interest. The strategies include germline and inducible cell typespecific knockdowns, which depending on the molecular switch applied can be either permanent (Cre/ loxP) or reversible (tetO/tTA). For reliable expression the shRNAs of interest are knocked into a pre-engineered Rosa26 docking site by recombinase mediated cassette exchange (RMCE). ES cells expressing the shRNA of interest can then be used to generate shRNA transgenic mice. The high efficiency of RMCE in ES cells enables the fast production of knockdown mice for in vivo functional analysis. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Silencing of gene expression by RNA interference (RNAi) is used as standard tool for functional genomics in cultured mammalian cells. RNAi is a sequence-specific gene silencing process that occurs at the messenger RNA (mRNA) level. In invertebrate cells, long double stranded RNAs (dsRNA), which are processed into short interfering RNAs (siRNA) by the ribonuclease Dicer, induce efficient and specific gene silencing. In mammalian cells, long dsRNAs (>30 bp) elicit an interferon response resulting in the global inhibition of protein synthesis and non-specific mRNA degradation. However, short dsRNAs (<30 bp) trigger the specific knockdown of mRNAs in mammalian cells without interferon activation [1]. Such synthetic siRNAs can be introduced into cultured cells and induce a transient knockdown of target genes. Alternatively, RNA polymerase III driven expression vectors enable the permanent production of small dsRNAs in mammalian cells [2,3]. Such vector derived transcripts are designed to contain an antisense region, complementary to a selected mRNA sequence, and the corresponding mRNA sense region. These transcripts fold into a stem-loop structure and form short hairpin RNAs (shRNAs) that are processed by Dicer in a similar way as siRNAs. Expression vectors for shRNAs ⇑ Corresponding author. Fax: +49 0 89 3187 3099. E-mail addresses: [email protected] (A. Kleinhammer), [email protected] (W. Wurst), [email protected] (R. Kühn). 1 Fax: +49 0 89 3187 3099. 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.12.015
can be stably integrated into the genome and allow permanent gene silencing in cell lines and organisms. Like in cultured cells gene silencing can be transiently induced in mice by delivering siRNA to somatic tissues. Direct siRNA delivery is frequently used in experimental settings requiring only a short term knockdown of gene function [4], e.g. to study and the replication of Hepatitis C virus in the liver [5]. In clinical studies, local delivery of siRNAs have already proved of value as disease gene inactivating drugs [6–9]. Alternatively, permanent gene silencing can be achieved in mice or rats by integrating shRNA transgenes into the genome. The efficiency of gene silencing in transgenic mice can be as high as 90% and sometimes even higher. In several instances, corresponding knockdown and knockout phenotypes were compared and found to be identical or very similar [4,10,11]. As compared to somatic siRNA delivery, for which the uptake rate is critical, transgenic shRNA is expressed in all cells of the organism. shRNA transgene delivery to mice can be achieved by pronucleus injection, viral embryo infection or targeted insertion (knock-in) into the ES cell genome [11–14]. In this article we summarize our approach for a streamlined production of shRNA transgenic mice using targeted transgenesis in ES cells [10,13,15,16]. This approach involves the insertion of shRNA expression vectors into the transcriptionally active Rosa26 locus of ES cells by recombinase mediated cassette exchange (RMCE) followed by the generation of knockdown mice. The targeted insertion of shRNAs into the Rosa26 locus ensures reproducible and ubiquitous transgene expression. A single shRNA vector copy expressed from the Rosa26 locus is sufficient for
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pEx-H1tetO-CAG-tetR pRMCE-II pCAG-C31Int pRosa26-50 probe
constitutive, body-wide gene silencing [15]. However, tissuespecific knockdowns require conditional vectors equipped with a molecular switch. Most frequently this switch is provided by the Cre/loxP recombination system enabling both a spatial and temporal control of shRNA expression [13]. Alternatively the tetO/tTA system can be used to reversibly induce gene silencing in a temporally and spatially restricted manner [17].
Plasmid sequence information can be found at: http://www. rnai.ngfn.de/index_4.htm.
2. Materials
2.2. Cell lines
2.1. Plasmids
ES cell line IDG26.10-3 ES cell line IDG3.2 Feeder cells: G418 resistant murine embryonic fibroblasts Plasmids and cell lines are available from the authors
pbs-U6 pNeb-lox-stop-lox
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attB
RMCE plasmid donor allele
attB neo
U6 shRNA
C31 Integrase PCR: 550 bp
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hyg1
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M C R EE os S a c W 26. ell 10 cl T ES E on ce S c e lls el ls
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Southern blot analysis
RMCE allele - 14 kb WT allele - 11 kb
Acceptor allele - 4.5 kb
Fig. 1. Genomic integration of shRNA vectors by RMCE into the Rosa26.10 allele of ES cells. (A) An shRNA expression cassette and a neomycin resistance gene (neo), flanked by two attB recognition sites for C31 Integrase, are located on a donor plasmid that is transfected into Rosa26.10 ES cells. (B) The Rosa26 acceptor locus in IDG3.2 Rosa26.10 ES cells contains a hygromycin resistance coding region flanked by two attP recognition sites for C31 Integrase. PCR genotyping can be performed with the primer pair hyg1/ hyg2 resulting in a 550 bp PCR product. Using the Rosa-50 genomic hybridization probe and EcoRV (E) digestion, the acceptor allele is recognized as a 4.5 kb band. (C) Upon C31 Integrase mediated recombination of both pairs of attB and attP sites the hygromycin resistance gene becomes exchanged by recombinase mediated cassette exchange (RMCE) against the shRNA expression unit. ES cell clones habouring RMCE alleles can be selected by the neo resistance gene that is expressed from the pgk promoter within Rosa26. Recombined ES cell clones can be identified by PCR genotyping using the primer pair pgk/neo that amplifies a 280 bp fragment. Using the Rosa-50 genomic hybridization probe and EcoRV (E) digestion, the RMCE allele is recognized as a 14 kb band. (D) Southern blot analysis of genomic DNA derived from wildtype ES cells, Rosa26.10 acceptor ES cells and an ES cell clone habouring a RMCE (shRNA) allele. Using the Rosa-50 genomic hybridization probe (B, C) and EcoRV digestion the Rosa26 wildtype allele exhibits a 11 kb band, as compared to a 4.5 kb band derived from the Rosa26.10 acceptor allele and a 14 kb band derived from the Rosa26.10 RMCE (shRNA) allele.
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2.3. Primers Neo: (50 -GTT GTG CCC AGT CAT AGC CGA ATA G-30 ), Pgk (50 -CAC GCT TCA AAA GCG CAC GTC TG-30 ), Hyg-1 (50 -GAA GAA TCT CGT GCT TTC AGC TTC GAT G -30 ), Hyg-2 (50 -AAT GAC CGC TGT TAT GCG GCC ATT G -3’), Rosa-50 (50 -CGT GTT CGT GCA AGT TGA GT30 ), Rosa-30 (50 -ACT CCC GCC CAT CTT CTA G-30 ). 2.4. Reagents Doxycycline: Doxycycline hyclate (Sigma). Dissolve in water (1 mg/ml) and store in single-use aliquots at 20 °C. G418: Geneticin solution with an active concentration of 50 mg/ mL (Invitrogen). Store in single-use aliquots at 20 °C. Gelatine: 0.1% gelatine in water, autoclaved. 3. Description of method 3.1. Overview We describe here three RNAi approaches in transgenic mice to control gene knockdown by RNAi that can be applied to any
transcript of interest. These are: (i) constitutive and body-wide gene silencing, (ii) temporally and spatially controlled gene silencing based on Cre/loxP recombination and (iii) temporally controlled gene silencing based on the tetR/O system. In each case a shRNA expression construct is stably integrated into the genome of ES cells and subsequently transferred to the mouse germline. In these constructs the shRNA of interest is expressed from an RNA Polymerase III promoter. To obtain the double-stranded RNA configuration required for short hairpin RNA formation from single-stranded RNA we link a 19–22 bp ‘sense’ siRNA sequence to its complementary ‘antisense’ sequence via an 8 bp loop region and combine it with a poly T termination signal (Fig. 2A). Upon transcription the complementary RNA strands hybridize to form dsRNA that is required for Dicer recognition and gene silencing. The easiest way to obtain a clonable DNA fragment yielding a sense-loop-antisense configuration is by annealing of two complementary oligonucleotides shA and shB of about 60 bp. Oligonucleotides shA and shB are flanked by restriction enzyme recognition sequences that form restriction enzyme compatible overhangs after hybridization (Fig. 2A). For stable integration of an shRNA expression vector into the ES cell genome we use recombinase mediated cassette exchange (RMCE). Fig. 1 illustrates the C31Int/attB/attP mediated cassette
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Fig. 2. Generation of constitutive shRNA vectors for RMCE. (A) shRNA vectors are designed for U6 promoter driven expression in such a way that a 19–22 bp ‘sense’ siRNA sequence is linked to its complementary ‘antisense’ sequence by an 8 bp loop region and followed by a poly T termination signal. The corresponding single-stranded RNA transcript will form a double-stranded short hairpin (sh) RNA through self-hybridization. The shRNA sequence is obtained by designing two self-complementary oligonucleotides whose hybridization results in a cloneable fragment with restriction enzyme compatible overhangs. The green and red arrows must be replaced by the sense and antisense target gene sequence. The required G at position +1 is part of the vector so that the first base of oligonucleotide shA represents the second transcribed nucleotide (+2) of the shRNA. (B) Cloning scheme for the ligation of hybridized oligonucleotides shA + B into the vector pbs-U6 resulting in a vector for shRNA expression. AsiSi and Sfil digestion allows for the isolation of a 0.35 kb fragment that contains the shRNA expression cassette and is used in the next cloning step (C). (C) Cloning of constitutive shRNA vectors for RMCE in IDG26.10-3 ES cells. Plasmid pRMCE-II that is compatible for RMCE is opened with AsiSI and SfiI and ligated with the 0.35 kb fragment from pbs-U6-shRNA (B). The resulting pRMCE-U6-shRNA plasmid can be used for genomic integration of the shRNA construct in IDG26.10-3 ES cells.
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stable knockdown ES cell lines. Moreover, at least two transgenic mouse lines expressing two different shRNAs against the same target are required to rule out off-target effects. Mouse lines are deemed safe for further analysis if the knockdown phenotypes of the two shRNA mouse lines are similar [22,23]. Below we give step-by-step instructions for the production of constitutive and inducible shRNA knockdown ES cell lines and mice.
exchange at a pre-engineered Rosa26 docking site [13]. As mentioned above, Rosa26 is a ubiquitously transcribed locus in ES cells insuring stable and ubiquitous expression of any knocked-in transgene. Since shRNA donor cassettes include a promoterless neomycin resistance gene (neo) which is activated only after correct cassette exchange at the Rosa26 acceptor locus (Fig. 1C), RMCE positive cell lines can be conveniently selected in G418. A more detailed description of the C31Int/attP/attB site-specific recombination system can be found Brown et al. and Monetti et al. (this issue, pages 372 and 380, respectively). After stable integration, vectors expressing shRNAs constitutively will directly induce gene silencing. A more detailed description of the induction mechanisms can be found elsewhere [18,19]. For the conversion of constitutive shRNA vectors into Cre-inducible vectors, a loxP-stop cassette which disrupts its function is inserted into a unique HindIII restriction site engineered into the shRNA loop region (Fig. 3). Given the diversity of the already existing Cre and Cre-ER transgenic driver lines (see Smedley et al., this issue, page 411, [20]) the Cre inducible shRNA transgenes enable detailed gene analysis in the context of an entire organism. Unlike the Cre/ loxP system, the tet repressor/tet operator (tetR/O) switch is not dependent on enzyme-based DNA rearrangement but the small synthetic compound doxycycline (dox). In the absence of dox the tetracycline repressor (tetR), expressed from a constitutive promoter, binds to the tet operator (tetO) of a modified H1 promoter and blocks shRNA transcription (Fig. 4B). Exposure to dox dissociates the tetR from tetO which induces shRNA expression (Fig. 4B) [19]. Thus, gene silencing is induced by dox addition [17,21]. By following the protocols described below, constitutive and conditional knockdown ES cell lines can be obtained in 2 months and the corresponding knockdown mouse lines in additional 4 months. As a rule, the gene silencing performances of five predicted siRNAs per target need to be evaluated in ES cells before creating
3.2. Constitutive knockdown 3.2.1. Cloning of constitutive shRNA vectors 1. Select five siRNA target sequences for your gene of interest with siRNA prediction tools. For the constitutive and the Cre-inducible knockdown vectors the U6 gene promoter is used. Make sure to use Guanine as the first nucleotide of the siRNA sequence because it is required for efficient transcription from the U6 promoter. Free siRNA prediction programs are available at the web pages of Ambion, Invitrogen or Qiagen and the University of Tokyo (http://genomics.jp/sidirect/index.php? type=fc), University of Vienna (http://rna.tbi.univie.ac.at/cgibin/RNAxs) and the Whitehead Institute (http://jura.wi.mit.edu/bioc/siRNAext/home.php). 2. Design a pair of complementary oligonucleotides shA and shB for each siRNA target sequence and follow the design shown in Fig. 2A. The purchase of expensive phosphorylated oligonucleotides is unnecessary as the vector ends remain phosphorylated after the following ligation reaction. 3. Anneal complementary oligonucleotides shA/B (1 lg/ll) by diluting 1 ll shA and 1 ll shB in 98 ll TE and incubating at 100 °C for 5 min. Allow the annealing reaction to cool down slowly to room temperature within 20 min.
A AsiSI
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U6
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conditional shRNA Vector (loxP)
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1.1.open withAsiSI AsiSI + SfiI open with + SfiI ligate with kbkb AsiSI-SfilI 2.2.ligate with1.2 1.2 AsiSI-SfilI fragment from pbs-U6-lox/loxfragment from pbs-U6-lox/loxshRNA shRNA attB
pRMCE-U6lox/lox-shRNA
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conditional shRNA Vector (loxP) for RMCE
Fig. 3. Generation of conditional Cre/loxP shRNA vectors for RMCE. (A) Constitutive shRNA vectors (pbs-U6-shRNA, Fig. 2) can be turned into Cre-incucible conditional shRNA vectors by introduction of a removable loxP-flanked stop cassette (863 bp MlyI fragment from pNEB-lox-stop-lox) into the unique HindIII restriction site within the loop region of the shRNA construct. The 1.2 kb conditional shRNA expression cassette can be isolated from pbs-U6-lox/lox-shRNA after digestion with AsiSI and SfiI. (B) Cloning of Cre-incucible conditional shRNA vectors for RMCE in IDG26.10-3 ES cells. Plasmid pRMCE-II that is compatible for RMCE is opened with AsiSI and SfiI and ligated with the 1.2 fragment from pbs-U6-lox/lox-shRNA (A). The resulting pRMCE-U6-lox/lox-shRNA plasmid can be used for genomic integration of the shRNA construct into IDG26.10-3 ES cells.
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A loop
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pEx-H1-tetOshRNA-CAG-tetR
attB
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Dox-inducible shRNA vector for RMCE
shRNA
CAG
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- dox tetR + dox:
Fig. 4. Generation of conditional tetR/O shRNA vectors for RMCE. (A) Two self-complementary oligonucleotides shA/B including the shRNA sequence are designed and hybridized resulting in a cloneable DNA fragment with an BsaBI compatible 50 end as this enzyme produces blunt ends. Additionally, the fragment is AscI compatible due to its overhang at the 30 end. Green and red arrows are meant to be replaced by target gene sense and antisense sequences respectively. The loop region contains a unique HindIII site and ‘‘TTTTTT’’ serves as Pol III termination signal. (B) Cloning instructions for the generation of a conditional tetR/O shRNA vector by ligation of the annealed oligonucleotides shA/B into the vector pEx-H1-tetO-CAG-tetR opened with BsaBI and AscI. The generated vector pEx-H1-tetO-shRNA-CAG-tetR provides all necessary elements of the tetR/O system. As the vector contains in addition attB sites and a promoterless neomycin resistance gene it can be used directly for RMCE. In the absence of dox the tetracycline repressor (tetR), expressed from the ubiquitous active CAG promoter, binds to the tet operator (tetO) of the H1-tetO promoter and blocks shRNA transcription. Exposure to dox dissociates tetR from tetO and thereby activates the H1 promoter resulting in the production of shRNA.
4. Digest the pbs-U6 plasmid DNA with BseRI and BamHI. Ligate 2 ll hybridization reaction (step 3) with 100 ng opened pbsU6 in a 10 ll standard ligation reaction at 16 °C overnight (Fig. 2). Redigest the ligation with BamHI to eliminate selfligated empty vector. 5. Transform the ligation reaction into Escherichia coli cells (we use DH5a), and digest miniprep DNA with PstI and HindIII. Correctly ligated clones show two bands of 310 bp and 2.9 kb. Plasmids without insert show only the 2.9 kb vector band. M13 forward and M13 reverse primers can be used for sequence confirmation. 6. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) with one confirmed clone for each of the 5 predicted target sequences and use these plasmids in the experiments. 3.2.2. Pretesting of knockdown efficiency by transient transfection into ES cells 1. For pretesting the shRNA knockdown efficiency in vitro electroporate the five plasmids individually into IDG3.2 ES cells grown on gelatine coated plates. For transfection use 40 lg circular plasmid DNA to electroporate 4 106 ES cells in 0.8 ml PBS with a 0.4 cm BioRad cuvette (we use the BioRad Genepulser Xcell set at 330 V and 3 ms time constant). Plate transfected cells into a 10 cm gelatine coated culture dish and refresh medium at the next day. 2. Harvest cells two days after electroporation and determine the expression level of your target gene by Western blotting or real time PCR. 3. Select the two best performing shRNA vectors yielding at least a 70% knockdown of the target protein/mRNA and use these for creating stable ES cell lines.
3.2.3. Stable integration of shRNA vectors into ES cells by RMCE 1. The U6-shRNA expression cassette is ligated into the pRMCE-II vector that provides elements (neomycin resistance, attB sites) required for RMCE into Rosa26. Digest the pbs-U6-shRNA plasmid with AsiSI and SfiI and the pRMCE-II vector with AsiSI and SfiI and dephosphorylate the ends (Fig. 2C). Ligate the 350 bp AsiSI-SfiI U6-shRNA fragment derived from pbsU6-shRNA into the AsisSI/SfiI pRMCE-II vector (4.3 kb). 2. Transform into E. coli cells, prepare minipreps and identify correct clones by digesting with SalI and HindIII. Correctly ligated clones show two bands of 1.6 kb and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. M13 forward, M13 reverse and bpA-for primers can be used for sequence confirmation. 3. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) and use these two generated plasmids for RMCE in ES cells. 4. Co-electroporate 25 lg (circular) DNA of each plasmid from step 3. together with 25 lg (circular) DNA of the C31 Integrase expression vector pCAG-C3Int into IDG26.10-3 ES cells grown on feeder layers. For electroporation of 50 lg plasmid DNA use 4 106 ES cells in 0.8 ml PBS with a 0.4 cm BioRad cuvette (we use the BioRad Genepulser Xcell set at 300 V and 2 ms time constant). Plate transfected cells into two 10 cm culture dishes containing embryonic fibroblasts as feeder layer. 5. On day 2 after transfection add G418 (140 lg/ml) to the culture medium. Keep in selection for 8 days by changing medium daily. 6. On day 9: Pick 12 (or more) colonies per RMCE experiment. Expand cells on feeders for freezing and on gelatine for DNA isolation.
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7. Extract genomic DNA by standard procedures [24] and identify positive clones by PCR or Southern blotting as described below. 3.2.4. Identification of RMCE positive ES cell clones 1. For PCR identification of RMCE positive ES cell clones use the neo and pgk primers listed in Section 2.3 (annealing temperature: 65 °C; correct product size 280 bp). Note that feeder cells containing a pgk-neo resistance gene will yield an additional amplification product of 160 bp. Incorrectly recombined clones will still contain the hygro gene which can be checked by using the Hyg-1 and Hyg-2 primers listed in Section 2.3 (annealing temperature 65 °C, product size 550 bp). Note that using feeders harbouring an additional hygro resistance gene precludes this screening procedure. 2. Southern blot verification of correct clones is mandatory for exclusion of partially recombined alleles or chromosomal rearrangements. To distinguish the wildtype and RMCE Rosa26 alleles cleave the genomic DNA with EcoRV and use the 450 bp EcoRI fragment from plasmid pRosa-50 probe as hybridization probe. The wildtype Rosa26 locus is identified by a band of 11.5 kb (Fig. 1D). The parental IDG26.10-3 ES cells show an additional band of 4.5 kb derived from the modified Rosa26 allele before RMCE (Fig. 1D). Positive RMCE clones show an additional band of 14 kb due to the integration of the U6-shRNA and neo gene cassettes (Fig. 1D). Usually 40% of the neomycin resistant clones underwent complete RMCE. 3. Evaluate gene silencing efficiency of confirmed RMCE clones by subjecting aliquots to Western Blot and/or RT PCR analysis as described in Section 3.2.2. 4. Use confirmed clones for generating mouse lines by blastocyst injection or tetraploid embryo aggregation as described in [25]. 3.3. Cre-inducible conditional knockdown 3.3.1. Cloning of Cre/loxP conditional shRNA vectors 1. Open the vector pbsU6-shRNA (see Section 3.2.1) with HindIII, fill in ends and dephosphorylate the 50 -overhanging ends using standard procedures [24] (vector fragment size is 3.3 kb). 2. Prepare the 863 bp lox-STOP-lox cassette from the plasmid pNEB-lox-stop-lox plasmid by digesting with MlyI. 3. To generate plasmid pbs-U6-lox/lox-shRNA, ligate the fragment containing the lox-STOP-lox cassette from step 2 into the pbsU6-shRNA plasmid from step 1 and eliminate religated vector background by digesting with HindIII. 4. Transform the ligation product into E. coli cells and and screen minipreps for inserts by digesting with EcoRI and SacII. Correctly ligated clones show an 850 bp band for the insert and a 3.4 kb vector band. Inversely oriented inserts produce 150 bp and 4 kb bands. Clones without insert show only the vector band. Use M13 forward and M13 reverse primers for sequence confirmation. 5. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) for one confirmed clone. 6. Test the functionality of the lox-STOP-lox cassette as described below. 3.3.2. In vitro lox-STOP-lox cassette excision and testing shRNA knockdown efficiency in ES cells 1. Delete the lox-stop-lox cassette in vitro by incubating 1 lg conditional shRNA plasmid DNA with 4 Units of Cre recombinase (New England Biolabs) in a total volume of 50 ll 1x Cre buffer at 37 °C for 30 min. Inactivate the recombinase for 10 min at 70 °C.
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2. Eliminate non-recombined plasmids by EcoRI digestion. 3. Transform the reaction into E. coli cells and confirm recombination by digesting miniprep DNA with XbaI. Cre recombined plasmids that lost the stop cassette produce two bands of 460 bp and 2.9 kb. Nonrecombined plasmids produce bands of 1.3 kb and 2.9 kb. After sequence confirmation using the M13 forward and reverse primers prepare a Maxiprep (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) from one confirmed clone. 4. Pretest the activated shRNA plasmids by transiently transfecting the Maxiprep DNA obtained in step 3 into ES cells as described in Section 3.2.2. 5. Choose these constructs in the conditional state for ligation into RMCE vectors and subsequent stable transfection into ES cells by recombinase mediated cassette exchange. The knockdown efficiency of stably integrated vectors should be tested in ES cells as described for constitutive vectors in Section 3.2.4, step 3 with the additional necessity of activating the integrated vectors by Cre before testing them. 3.3.3. Stable integration of Cre/loxP conditional shRNA vectors into ES cells by RMCE For construction of Cre-inducible shRNA vectors compatible for RMCE follow the protocol for the construction of constitutive shRNA RMCE vectors (Section 3.2.3). Here we point out only those steps that differ to the protocol in Section 3.2.3, step 1: Ligate the U6-shRNA-lox-stop-lox fragment (1.2 kb) into the opened pRMCEII vector (4.3 kb). Section 3.2.3, step 2: Identify correct clones by plasmid digestion with SalI and EcoRI. Correctly ligated clones show two bands of 1.7 kb and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. Section 3.2.4, step 2: The wildtype Rosa26 locus can be identified by a band of 11.5 kb (Fig. 1D). The parental IDG26.10-3 ES cells show an additional band of 4.5 kb derived from the modified Rosa26 allele before RMCE (Fig. 1D). Positive RMCE clones show an additional band of 15 kb due to the integration of the U6-shRNA-lox-stop-lox and neo gene cassettes. Section 3.2.4: step 4, Use only confirmed ES cell clones for generating mouse lines by blastocyst injection or tetraploid embryo aggregation. Resulting mice need to be mated to Cre deleter strains for knockdown activation. 3.4. Doxycycline inducible knockdown This strategy employs vector pEx-H1tetO-CAG-tetR consisting of a tetO modified H1 promoter driving shRNA expression, a codon optimized tetR gene controlled by a constitutive CAG promoter and a promoterless neo resistance coding region. These elements are flanked by attB sites to enable RMCE (Fig. 4B). 3.4.1. Cloning of conditional tetR/O shRNA vectors 1. Select five siRNA target sequences for your gene of interest as described in Section 3.2.1. siRNA sequences may start with any nucleotide since in this approach the H1 and not the U6 promoter is used. 2. Design and anneal shA and shB oligonucleotides for each selected mRNA target sequence as described in Section 3.2.1, steps 1–3 except that BsaBI and AscI compatible ends are added (Fig. 4A). 3. Digest the plasmid pEx-H1tetO-CAG-tetR with BsaBI and AscI (Fig. 4B) and ligate the double stranded shA/B DNA to the vector. Redigest the ligation with BsaBI and AscI to remove the self-ligated empty vector. 4. Transform the ligation product into E. coli cells (we use DH5a) and digest miniprep DNA with PstI and HindIII. Correctly ligated clones should yield 4 bands of 0.2 kb, 1.0 kb, 2.8 kb and 3.3 kbs.
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Empty plasmids should yield only 3 bands of 0.2 kb, 1.0 kb and 6.1 kbs. M13 forward and reverse primers can be used for sequence confirmation as described in Section 3.2.4, step 3. 5. Prepare Maxipreps (100 ml LB culture, Quiagen Plasmid Maxi kit, Quiagen) with one confirmed clone for each of the five predicted target sequences. 3.4.2. Pretesting of tetR/O shRNA vectors by transient transfection into ES cells Follow the steps described in Section 3.2.2 using 1 lg/ml dox in the culture medium to induce the knockdown. Select the two best performing shRNA vectors (>70% KD activity of the target) for RMCE. 3.4.3. Stable integration of tetR/O shRNA vectors into ES cells by RMCE 1. The pEx-H1tetO-shRNA-CAG-tetR plasmids generated in Section 3.4.1, step 6 can be directly used for RMCE in IDG26.10-3 ES cells. For RMCE follow steps 4–7 of Section 3.2.3. Identify RMCE positive subclones by PCR and Southern blot as described in Section 3.2.4. The wildtype Rosa26 locus can be identified by a band of 11.5 kb (Fig. 1D). The parental IDG26.10-3 ES cells show an additional band of 4.5 kb derived from the modified Rosa26 allele before RMCE (Fig. 1D). Positive RMCE clones exhibit a 17.7 kb band corresponding to the cassette exchange allele in addition to the 11.5 kb wild type allele. 2. Evaluate gene silencing efficiency as described in Section 3.2.4, step 3 using western blotting and/or or RT-PCR. For knockdown induction add 1 lg/ml doxycycline to the culture medium and set up and perform two negative controls in parallel. These are: (i) tetR/O shRNA positive cells kept in dox-free medium and IDG26.10-3 parental cells exposed to 1 lg/ml dox. The first control serves for estimating the extent of dox induction whereas the second reflects vector leakiness. 3. In mice knockdown activation is achieved by supplying the drinking water with 2 mg/ml dox and 10% sucrose. Light protection of dox containing water bottles is required; the water should be replaced every other day to maintain constant blood levels of dox. 4. Concluding remarks – how to use KD ES cell lines and mice for extracting function from the mammalian genome The shRNA vector approach described in this article enables body-wide and cell type-specific gene silencing in most tissues of adult mice. Particulary in the brain mRNA and protein levels can be reduced up to 90% with this technology [10,13,15,16]. However, the major factor for successful gene silencing in vivo is the preselection of highly effective shRNA sequences by in vitro siRNA screening. Compared to the generation of knockout mice by gene targeting the shRNA approach is technically less demanding and does not require homozygosity for the functional analysis of recessive genes. The knock-in insertion of shRNA vectors into a Rosa26 universal docking site enables the production of transgenic mice with reproducible shRNA expression patterns. Moreover, the generation of knockdown mice is relatively fast – a first group of adult mutants can be obtained within a period as short as 11 months [17,26,27]. The development of genetic mouse models that mimic human disease conditions to elucidate pathogenic mechanisms is the most direct application of in vivo RNAi [10,28]. For many complex diseases an increasing number of risk alleles and modifiers are being discovered by genome wide association studies and more recently also by the analysis of copy number variation [29]. Accordingly, the targeted modification of several genes within the same organism is highly desirable. Standard knockout technology can not be easily scaled up to more than two genes. Since gene silencing is indepen-
dent of a specific genomic target sequence, RNAi technology provides the means for simultaneously manipulating a significantly larger number of genes. For example, a single Rosa26 shRNA allele conferring a single gene knockdown can be easily combined with a second Rosa26 knockdown allele by simple breeding. If each of these alleles would be equipped with shRNAs directed against two different targets simple breeding could achieve knockdown of up to four target genes in the same organism by using only two transgenic loci that can be further freely combined with other knockdown units. Further transgenic RNAi applications include the validation of proteins as drug targets to predict the therapeutic potential of loss-of-function phenotypes [30–32]. Finally, modified siRNAs can be directly used as drugs and have proved successful in several clinical trials [6], most notably in the treatment of macular degeneration where the knockdown of vascular endothelial growth factor (VEGF) gene by local application of modified siRNAs significantly ameliorated the disease [33]. Acknowledgments This work has been funded by the Volkswagen Foundation and the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (FKZ: 01GR0404 and 01GS0858). We thank H. von Melchner for critical reading of the manuscript. References [1] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Nature 411 (2001) 494–498. [2] T.R. Brummelkamp, R. Bernards, R. Agami, Science 296 (2002) 550–553. [3] P.J. Paddison, A.A. Caudy, E. Bernstein, G.J. Hannon, D.S. Conklin, Genes Dev. 16 (2002) 948–958. [4] R. Kuhn, S. Streif, W. Wurst, Handb. Exp. Pharmacol. (2007) 149–176. [5] Q. Pan, H.W. Tilanus, H.L. Janssen, L.J. van der Laan, Expert Opin. Biol. Ther. 9 (2009) 713–724. [6] A.K. Vaishnaw, J. Gollob, C. Gamba-Vitalo, et al., A status report on RNAi therapeutics, Silence 1 (2010) 14. [7] D.M. Dykxhoorn, D. Chowdhury, J. Lieberman, Adv. Exp. Med. Biol. 615 (2008) 299–329. [8] D.M. Dykxhoorn, J. Lieberman, Cell 126 (2006) 231–235. [9] K. Tiemann, J.J. Rossi, EMBO Mol. Med. 1 (2009) 142–151. [10] S. Delic, S. Streif, J.M. Deussing, et al., Genes Brain Behav. 7 (2008) 821–830. [11] T. Kunath, G. Gish, H. Lickert, N. Jones, T. Pawson, J. Rossant, Nat. Biotechnol. 21 (2003) 559–561. [12] H. Hasuwa, K. Kaseda, T. Einarsdottir, M. Okabe, FEBS Lett. 532 (2002) 227–230. [13] C. Hitz, W. Wurst, R. Kuhn, Nucleic Acids Res. 35 (2007) e90. [14] D.A. Rubinson, C.P. Dillon, A.V. Kwiatkowski, et al., Nat. Genet. 33 (2003) 401– 406. [15] J. Seibler, B. Kuter-Luks, H. Kern, et al., Nucleic Acids Res. 33 (2005) e67. [16] P. Steuber-Buchberger, W. Wurst, R. Kuhn, Genesis 46 (2008) 144–151. [17] J. Seibler, A. Kleinridders, B. Kuter-Luks, S. Niehaves, J.C. Bruning, F. Schwenk, Nucleic Acids Res. 35 (2007) e54. [18] M. Lewandoski, Nat. Rev. Genet. 2 (2001) 743–755. [19] M. Wiznerowicz, J. Szulc, D. Trono, Nat. Methods 3 (2006) 682–688. [20] A. Nagy, L. Mar, G. Watts, Methods Mol. Biol. 530 (2009) 365–378. [21] M. van de Wetering, I. Oving, V. Muncan, et al., EMBO Rep. 4 (2003) 609–615. [22] E.M. Anderson, A. Birmingham, S. Baskerville, et al., RNA 14 (2008) 853–861. [23] A. Birmingham, E.M. Anderson, A. Reynolds, et al., Nat. Methods 3 (2006) 199– 204. [24] J. Sambrook, P. Macallum, D. Russell, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbour Press, Cold Spring Harbour, 2001. [25] S.W. Reid, L. Tessarollo, Methods Mol. Biol. 530 (2009) 269–285. [26] M.J. Herold, J. van den Brandt, J. Seibler, H.M. Reichardt, Proc. Natl. Acad. Sci. USA 105 (2008) 18507–18512. [27] K. Kotnik, E. Popova, M. Todiras, et al., PLoS ONE 4 (2009) e5124. [28] H. Zhou, B.H. Falkenburger, J.B. Schulz, K. Tieu, Z. Xu, X.G. Xia, Int. J. Biol. Sci. 3 (2007) 242–250. [29] L. Fugger, M.A. Friese, J.I. Bell, Nat. Rev. Immunol. 9 (2009) 408–417. [30] T. Christoph, G. Bahrenberg, J. De Vry, et al., Mol. Cell. Neurosci. 37 (2008) 579– 589. [31] R. Sacca, S.J. Engle, W. Qin, J.L. Stock, J.D. McNeish, Methods Mol. Biol. 602 (2010) 37–54. [32] R. Yang, G. Castriota, Y. Chen et al., Int. J. Obes. (Lond)., 2010, doi:10.1038/ ijo.2010.128. [33] L. Singerman, Retina 29 (2009) S49–S50.