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Generating mutant rats using the Sleeping Beauty transposon system
Methods 49 (2009) 236–242
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Methods journal homepage: www.elsevier.com/locate/ymeth
Generating mutant rats using the Sleeping Beauty transposon system Kazuhiro Kitada a, Vincent W. Keng b, Junji Takeda c,d, Kyoji Horie c,* a
Laboratory of Mammalian Genetics, Division of Genome Dynamics, Creative Research Initiative ‘‘Sousei”, Hokkaido University, North 10 West 8, Kita-ku, Sapporo 060-0810, Japan Masonic Cancer Center and Center for Genome Engineering, University of Minnesota, 420 Washington Avenue SE, Minneapolis, MN 55455, USA Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan d Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan b c
a r t i c l e
i n f o
Article history: Accepted 10 April 2009 Available online 3 May 2009 Keywords: Mutagenesis Transposon Sleeping Beauty Rat
a b s t r a c t The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 30 -rapid amplification of cDNA ends (30 RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The laboratory rat (Rattus norvegicus) remains the major animal model system inside the pharmaceutical and biomedical research industries largely because ‘‘size does matter”. Importantly, rat models tend to be disease based, generating more information about the actual disease of interest, rather than just being an animal model. Unfortunately, geneticists have long preferred the mouse model because of its smaller size, which simplified housing requirements, and the availability of many coat color and mutants exhibiting Mendelian patterns of inheritance. In addition, the availability of mouse embryonic stem (ES) cells for gene-specific characterization and knockout studies has made this model indispensable. Although the availability of rat transgenic technologies have allowed advances in the field of gene-specific analysis, the lack of rat ES cells has hampered any further progress in this field. Germline-competent rat ES cells has been reported recently and may revolutionize this field [1,2]. However, the robustness for large-scale generation of mutant rats remains to be tested. Chemical mutagenesis has been widely used in the rat [3], however, a large portion of induced mutations are point-mutation and thus not null alleles in most cases, and moreover, identifying the causative gene seems to be a challenging task by this method. * Corresponding author. Fax: +81 6 6879 3266. E-mail address: [email protected] (K. Horie). 1046-2023/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2009.04.010
Sleeping Beauty (SB) transposon system is a novel genetic tool developed around a decade ago [4]. It consists of two components: The SB transposon, which is excised and reinserted into other locations of the genome, and the SB transposase catalyzes this reaction. Since most of the vertebrate transposase genes are inactivated during evolution, the SB transposase was reconstructed by extensive manipulation of inactive transposase copies from the fish genome. The SB transposon contains inverted repeat/direct repeat (IR/DR) elements at either ends (Fig. 1a), which are essential sequence for the recognition and mobilization by the SB transposase. Recently, others and we have demonstrated the effectiveness of SB for large-scale germline mutagenesis in mice [5–7]. Analysis of the SB transposition sites revealed preference of transposition on the donor-site chromosome, which varied between 60% to 80% amongst SB transposon-transgenic lines, whereas remaining 20– 40% was distributed in various locations of the genome [5,6]. Many of the transpositions on the donor-site chromosome were clustered within 3–4 Mb from the donor site [5,6]. Others and we further applied the SB transposon system in rats, and succeeded in rapid generation of mutants [8,9]. In the present paper, we describe a protocol for the generation and analysis of the mutant rats based on the SB transposon vectors developed in our laboratory [8]. It should be noted that various transposon systems have been developed recently and some of them have been shown to be active in mammalian cells, such as Tol2 [10], PiggyBac [11], Minos [12], and Frog Prince [13]. Although
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a
SB transposon
Transposon vector
IR/DR
IR/DR SA IRES LacZ pA
Promoter trap system for X-gal detection
Transposase vector
b
E1
Plasmid backbone
CAG GFP SD
PolyA trap system for noninvasive GFP detection
CAG SB pA
E2
E3 SA IRES LacZ pA (A)n
Truncated endogenous protein
c
SB
X
E4
CAG GFP SD (A)n
GFP
ß-gal
Gfp GFP
SB Transposase Tg rat
Transposon Transposon TgTgrat rat
SB GFP Gfp
X
WT
Double Tg rat "seed rat"
Wild-type rat
Screening for GFP-positive rats
Gfp GFP No gene-trapped event
Bright-field
Fluorescence
Gfp GFP Gene-trapped event
DNA
RNA
LM-PCR (Fig. 2)
3’ RACE (Fig. 3)
Production of progeny, Intercross for homozygous mutant generation, & phenotypic analysis
Fig. 1. Overview of the mutagenesis strategy. (a) Structure of vectors used to generate transgenic lines. Transgenic rats bearing the transposon vector (top) and the transposase vector (bottom) were generated independently, and mated to generate doubly-transgenic rats as shown in (c). IR/DR is the recognition sequence of the SB transposase. The region flanked by IR/DRs is excised by the SB transposase and inserted into other locations of the genome. SA, splice acceptor; SD, splice donor; IRES, internal ribosome entry site; pA, polyA signal; CAG, cytomegalovirus enhancer/chicken beta-actin chimeric promoter; IR/DR, inverted repeat/direct repeat. (b) Scheme of the promoter- and polyA-traps. In this example, the SB transposon vector is inserted into the 2nd intron. The transcript from the inserted gene is trapped by the splice acceptor of the transposon vector. As a result, production of the endogenous protein is disrupted and beta-galactosidase from the LacZ gene is expressed, which can be used as a reporter for the expression profile of the mutated gene in various tissues. The GFP reporter is driven by the constitutively active CAG promoter and is expressed as a result of the splicing between vector-derived splice donor and the splice acceptor of the downstream exon. E, exon. (c) Breeding scheme for the generation of mutant progeny and an example of the screening for mutants based on GFP expression. The rat at the top of the photograph is GFP-positive and therefore presumably an insertional mutant, while the rat at the bottom is GFP-negative and therefore, a non-mutant.
their application for insertional mutagenesis in rat remains to be addressed, it is conceivable that some of them will have different characteristics compared with SB, such as different patterns of transposition distribution [14] and different rates of transposition events. Therefore, it is highly likely that these DNA-type transposable elements will complement SB as a mutagenesis tool. Most of the protocol described in this paper can be easily modified and applicable to other transposon systems by simple modification.
2. Description of method 2.1. Overview (Fig. 1) Vector structures and general mutagenesis scheme are shown in Fig. 1. The initial part is to generate two transgenic rat lines, one containing a donor concatemer of transposon gene-trap vec-
tors and the other containing a source of transposase (Fig. 1a). The transposon gene-trap vector concatemer source consists of a promoter-trap component and a polyA-trap component. The promoter-trap component consists of human BCL2 intron 2/exon3 splice acceptor (SA) [15], encephalomyocarditis virus internal ribosome entry site (IRES), the LacZ gene and rabbit beta-globin polyA signal (pA). The polyA-trap component contains the cytomegalovirus (CMV) enhancer/chicken beta-actin chimeric promoter (CAG promoter) [16], the GFP gene, and splice donor (SD) from the mouse Hprt gene exon 8/intron 8 region which is followed by the mRNA instability signal derived from the human granulocyte-macrophage colony-stimulating factor cDNA [15]. The source of transposase is made using the CAG promoter driving SB transposase. These lines are mated with each other to generate doubly-transgenic rats, and the doubly-transgenic rats are bred to wild-type rats (Fig. 1c). Transposition in the germline of the doubly-transgenic rats (‘‘seed rat” in Fig. 1c) will generate heterozy-
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gous mutant rats in the progeny. If transposon integration occurs within a gene, the promoter-trap component allows for the detection of LacZ staining, relying on the endogenous promoter (Fig. 1b). In addition, the polyA-trap component relies on the polyA signal of the endogenous gene to produce the GFP protein and fluorescence can be detected non-invasively (Fig. 1b and c). Mutated genes are identified with either of the two methods: (1) Linker ligation-mediated PCR (LM-PCR) (Fig. 2), and (2) 30 -rapid amplification of cDNA ends (30 RACE) (Fig. 3). The former method determines the exact vector insertion site at the nucleotide level. The latter method amplifies trapped transposon-endogenous exon sequence from mRNA. These methods are complementary to each other in identifying the insertion site of the transposon vector. The first approach is relatively simple and straightforward but prediction of the mutated gene is dependent on the rat genome annotation database, in which the mutated gene may not be mapped yet. The latter method may reveal novel transcripts, which are not yet annotated in the genome database, but the transposon insertion site could be unexpectedly distant from the trapped site. 50 RACE is another useful approach to determine novel transcripts and has been widely used in gene trap in mouse embryonic stem cells [17]. We use 30 RACE because we routinely use ear or tail clip as a source of RNA and the mutated gene under study may not be expressed there.
Transposition site
Transposition may occur in somatic cells of mutant rats when the SB transposase is not segregated. Our experience indicates that its frequency is not high enough to create confusion in the analysis of germline transpositoin sites. However, it is advisable to select for mutant rats that do not contain the SB transposase in order to avoid continuous germline transposition in the progeny. 2.2. Generation of transgenic rats containing the transposon and the transposase vectors 2.2.1. Consideration for vector construction For generation of transgenic rats, plasmid backbone sequence is usually removed from vector DNA prior to pronuclear injection. We followed this rule for the injection of the SB transposase vector. In contrast, we did not remove the plasmid backbone (pBluescript and neo gene cassette) for the injection of the transposon vector (Fig. 1a). These sequences are not required for the function of the transposon. However, our experience suggests that the presence of this region induces methylation of the transposon DNA and inactivate the CAG promoter [18]. As a result, GFP fluorescence from the donor site is prevented. In contrast, the CAG promoter is activated at the transposition site because the transposon is not flanked by the plasmid backbone any more. As a result, gene insertion events can be
Vector concatemer at the donor site
Transposon
Plasmid backbone Genomic sequence
X
IR/DR
X
X
IR/DR
IR/DR
Y
X
IR/DR
Digestion with enzyme “X” X
X
Y
X
X
Linker ligation Y
Linker
Digestion with enzyme “Y” Y
Y
1st PCR primers
1st PCR No amplification 2nd PCR primers 2nd PCR
Sequencing
A
G
T
C
Fig. 2. Linker ligation-mediated PCR (LM-PCR). Amplification of rat genomic sequence at transposition sites (left) and the elimination of undesired amplification products derived from the transposon vector donor concatemer (right). Genomic DNAs from GFP-positive rats (Fig. 1c) are digested with the enzyme ‘‘X”, a linker DNA is ligated, and transposition sites are amplified by nested-PCR using vector-specific primers and linker-specific primers. To avoid amplification of the vector concatemer at the donor site, linker-ligated DNAs are digested with the enzyme ‘‘Y” which is located just outside of the IR/DR. We usually use 4-base cutters for enzyme ‘‘X” and 6-base cutters for enzyme ‘‘Y”.
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SA IRES LacZ pA
CAG GFP SD AAAA------AA
GFP
Trapped sequence AAAA------AA
mRNA
DNA from step (1) Buffer (5) dNTP (10 mM) MgCl2 (50 mM) Primer mix* (10 lM each) Tfi DNA polymerase (Invitrogen) DW Total
1 ll 5 ll 0.5 ll 0.75 ll 0.5 ll 0.5 ll 16.75 ll 25 ll
Reverse transcription
mRNA
AAAA------AA
cDNA
TTTT----
Oligo-dT primer
* PCR primer pairs: 50 -AAGACCCATTTGCGACCAAGCTTTA-30 and 50 CCGAAGAACACCATCCCAACCGTGA-30 for SB transposase, and 50 -AA CGTCTATATCATGGCCGACAA-30 and 50 -TGGGGGTGTTCTGCTGGTAG TGGTC-30 for SB transposon lines, respectively. 3. Perform PCR reaction. PCR condition:
1st PCR 1st PCR primer TTTT----
1st PCR primer AAAA---TTTT---AAAA---TTTT---AAAA---TTTT---2nd PCR primers 2nd PCR
AAAA---TTTT---AAAA---TTTT---AAAA---TTTT----
Sequencing
A
G
T
C
Fig. 3. 30 -rapid amplification of cDNA ends (30 RACE). Total RNA from GFP-positive rats (Fig. 1a) are reverse transcribed with the oligo-dT primer, and trapped sequences are amplified by nested-PCR using GFP-specific primers and oligo-dTspecific primers. Abbreviations are as described in Fig. 1.
visualized as ‘‘green rats” (Fig. 1c). Furthermore, our in vitro study indicated that the DNA methylation and heterochromatinization of the transposon donor site significantly enhances SB transposition [19,20]. It should be noted that the enhancement of transposition is not necessarily seen in other transposon systems [14]. 2.2.2. Maintenance of transgenic rat lines It is important that transgenic lines harboring SB transposase and transposon vector should be maintained independently. Maintenance as a doubly transgenic line is not recommended because the structure and copy number of the donor concatemer may be altered due to germline transposition of the SB transposon. The PCR genotyping protocol of the SB transposase and the SB transposon lines that were reported in our study [8]. 1. Dilute genomic DNA to a 100 ng/ll concentration using distilled water (DW). 2. Set up the following PCR reaction:
94 °C 94 °C 55 °C 72 °C 72 °C 25 °C
2 min 30 s 1 min 45 s 10 min Hold
1 cycle 30 cycle 1 cycle
4. Separate PCR products in a 2–4% agarose gel containing ethidium bromide for visualization. PCR amplicon of 191-bp will be seen in transgenic lines harboring the SB transposase, and a 118-bp PCR product will be seen in transgenic lines harboring the SB transposon vector, respectively. 2.2.3. Selection for GFP-negative rat As mentioned above, it is expected that a plasmid backbone of the transposon vector should prevent GFP expression due to the highly methylated state of the transposon donor concatemer. However, some transposon vector-carrying transgenic lines do reveal detectable GFP expression even though no transposase-induced transposition had occurred. Such GFP-positive lines must be excluded from further analyses. We recommend that this step should be performed at G1 generation, and not at the founder generation, considering possible mosaicism is occasionally seen in the founder generation. Selection for GFP-negative rat can be performed as described in the Section 2.3.1. 2.2.4. Testing for the effect of homozygosity at the donor site It is reported that ‘‘local hopping” events are frequently observed for the SB transposon system [5–7]. Segregation between a transposed site and the transposon vector donor concatemer site may not be easily achieved by recombination in some cases. In order to precisely evaluate trapped gene events, it is essential to confirm that homozygosity of the donor concatemer site does not generate any overt phenotype before utilizing the transgenic line for further analyses. For this purpose, G2 offspring are generated by intercrossing G1 transgene-positive rats described earlier in Section 2.2.2. Identification of homozygotes in the G2 offspring can be achieved by FISH (fluorescence in situ hybridization) analyses or the classical testcross analyses [21]. Selected G2 homozygotes are then aged and examined carefully. 2.2.5. Screening of the SB transgenic lines The expression level of the SB transposase varies among transgenic lines and some lines may not exhibit any expression. Therefore, it is recommended to analyze the expression of the SB transposase in the male germline and identify appropriate transgenic lines. The standard methods such as immunohistochemistry,
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northern blotting or RT-PCR can be used. Antibody against SB transposase is commercially available from R&D Systems. 2.3. Generation and identification of mutant rats Transgenic lines harboring SB transposase and transposon vector are intercrossed and doubly-transgenic rats are selected as described in Section 2.2.2. Doubly-transgenic rats ‘‘seed rat” are then mated with wild-type rats. Transposition in the germline of these doubly-transgenic rats will generate heterozygous mutant rats in the progeny. Mutant rats are easily and rapidly selected by a non-invasive method using a fluorescent microscope (Fig. 1c). We usually use male doubly-transgenic rats because more breeding pairs can be set up compared with female ‘‘seed rats”. 2.3.1. Detection of GFP-positive mutant rats using fluorescent microscope As GFP signals are generally weak in our gene-trapped mutant rats, the use of a fluorescent microscope with high enough sensitivity is essential and a hand-held UV light source is not suitable for this purpose. We routinely use a SMZ800 fluorescent dissecting scope (Nikon) with a filter block P-FLGFP-B (460–500 nm band pass excitor filter and 510–560 nm barrier filter). With the detection of a weak GFP signal, it is recommended that screening be performed on neonates around 3-days of age. Delayed screening should be avoided as hair development occurs and the detection of GFP signal can become very difficult. 2.3.2. Frequency of mutation in progeny In our transposon system, approximately 11% of newborn rats from doubly-transgenic rats are GFP-positive, even though this frequency seems to vary amongst transposon vector lines. In our hands, it is determined that the transposition events in germ line cells occur with a rate of approximately one transposition event per spermatozoon. 2.3.3. Consideration for possible chromosomal rearrangement during transposition It should be noted that chromosomal rearrangement near the donor concatemer might arise during the transposition reaction [22]. Since the SB transposon has a preference to transpose near the donor site [5–7], there is a possibility that some of the transposon insertions are tightly linked to the rearranged region and cannot be segregated easily. This possibility should be kept in mind during phenotypic analyses because the observed phenotypes could be due to the chromosomal rearrangement and not the transposition sites. Therefore, mapping the location of the insertion mutations is a critical part of their ultimate usefulness and should be evaluated in the initial phase of the phenotypic analysis. 2.4. The mapping of insertion sites by LM-PCR (Fig. 2) 2.4.1. Isolation of genomic DNA Tail or ear clips from rats (‘‘green rat” in gene-trapped event in Fig. 1c) can be used as a source of genomic DNA. The protocol described below is based on the classical method involving phenol/ chloroform extraction and ethanol precipitation techniques. We have also used column-based commercial kits (e.g. DNeasy Blood & Tissue Kit, Qiagen) and insertion sites could also be determined seamlessly. Since amplification by the LM-PCR protocol is robust, most DNA isolation methods that provide DNA quality compatible with Southern analysis will be adequate for LM-PCR. 1. Lyse source material (e.g. 0.5 cm of tail clip) in 1.5 ml microtubes using a DNA extraction buffer containing proteinase K (500 ll of DNA extraction buffer [10 mM Tris–
2.
3.
4.
5. 6. 7. 8. 9. 10. 11.
HCl, 1 mM EDTA, 1 SSC, 1% SDS] with 10 ll of proteinase K [10 mg/ml stock, Merck] added) – incubate at 55 °C overnight with shaking. Centrifuge at 15,000 rpm for 5 min at 4 °C to pellet undissolved material – transfer liquid phase to a fresh microtube using pipette tips cut at the end to avoid genomic DNA shearing. Add equal volume (500 ll) of phenol:chloroform (1:1 ratio) – mix for a few min by gentle inversion (this step may be repeated if protein interface contamination occurs). Centrifuge at 15,000 rpm for 10 min at 4 °C – transfer aqueous phase to a fresh microtube using pipette tips cut at the end to avoid genomic DNA shearing. Add 0.7 volume (350 ll) of isopropanol– mix for a few min by gentle inversion. Centrifuge at 15,000 rpm for 10 min at 4 °C to pellet genomic DNA precipitate. Discard supernatant – wash with 500 ll 70% ethanol. Centrifuge at 15,000 rpm for 10 min at 4 °C to pellet genomic DNA precipitate. Discard supernatant – dissolve genomic DNA in 50–200 ll TE (Tris–HCl 10 mM; EDTA 1 mM). Leave overnight at 4 °C to fully dissolve genomic DNA in TE. Mix gently, spin down and obtain OD reading at 260/280 nm to determine concentration and purity (260/280 ratio should be greater than 1.8).
2.4.2. Linker-ligation and PCR 1. Dilute genomic DNA to a 10 ng/ll concentration using DW. 2. Digest 100 ng of diluted genomic DNA at 37 °C for 2–3 h in a final volume of 50 ll using an appropriate 4-base restriction enzyme (enzyme ‘‘X” in Fig. 2). It should not cleave between the transposon-specific primer (see below, indicated by arrowhead in Fig. 2) and the end of the IR/DR. In the case of our SB transposon vector, the following enzymes (New England Biolabs) can be used: i. AluI, ii. MboI, iii. HaeIII 3. Heat-inactivate restriction enzymes by incubating at the following temperatures for 20 min: i. AluI, MboI – 65 °C, ii. HaeIII – 80 °C. 4. Linkers (Splinkerettes [23]) are prepared by annealing the following oligonucleotide sequences together (10 lM each): Spl-top 50 -CGAATCGTAACCGTTCGTACGAGAATTCGTACGAGAATCGCTGTCC TCTCCAACGAGCCAAGG-30 with either SplB-blunt 50 -CCTTGGCTCG TTTTTTTTTGCAAAAA-30 to obtain the final Spl-blunt linker; or Spl-top with SplB-sau 50 -GATCCCTTGGCTCGTTTTTTTTTGCAAAAA30 to obtain the Spl-sau linker with 50 -protruding end. Combination of the cleavage end and the linker is as follows: i. AluI, HaeIII– use Spl-blunt, ii. MboI – use Spl-sau. Set up following ligation reaction (Takara DNA ligation kit ver. 1) and incubate at 16 °C for at least 3 h: Enzyme-digested genomic DNA Appropriate linker Buffer A Buffer B Total
2 ll 1 ll 12 ll 3 ll 18 ll
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5. Purify using Qiagen QIAquick PCR purification kit (using manufacturer’s instructions) – resuspend in 40 ll DW. 6. Digest each sample with a 6-base restriction enzyme (or any appropriate rare cutter enzyme, indicated by enzyme ‘‘Y” in Fig. 2) that cleaves the transposon vector backbone sequence just outside of the IR/DR but does not cleaves between the transposon-specific primer (see below, indicated by arrowhead in Fig. 2) and the end of the IR/DR. This step prevents amplification of the transposon vector donor concatemer site. In our SB transposon vector, KpnI is appropriate for this purpose. In case the donor site is segregated during the breeding process, this step can be omitted. Set up the following reaction and incubate at 37 °C overnight to accomplish complete digestion. 39 ll 5 ll 5 ll 1 ll 50 ll
Linker-ligated/purified DNA Appropriate buffer (10) BSA (10) Enzyme (in excess) Total
7. Purify using Qiagen QIAqucik PCR purification kit (using manufacturer’s instructions) – resuspend in 50 ll DW (may omit if sample quantity is large and can proceed directly to nested-PCR). 8. Nested-PCR. 8-1. 1st PCR reaction Set up following PCR reaction: 1 ll 5 ll 1 ll 1 ll 1 ll 0.25 ll 40.75 ll 50 ll
DNA from step (7) Buffer (10) dNTP (10 mM) T/DR2* (10 lM) Spl-P1** (10 lM) HotStarTaq (Qiagen) DW Total
* T/DR2 (transposon-specific primer): 50 -CTGGAATTGTGATACA GTGAATTATAAGTG-30 . ** Spl-P1 (linker-specific primer): 50 -CGAATCGTAACCGTTCGTAC GAGAA-30 . Transposon-specific primer is designed in the IR/DR region downstream of the polyA-trap component. Therefore, genomic sequence at the downstream of the transposon insertion site is amplified. PCR condition:
95 °C 95 °C 55 °C 72 °C 72 °C 25 °C
15 min 1 min 1 min 1 min 7 min Hold
1 cycle 30 cycle 1 cycle
T/BAL (transposon-specific primer): 50 -CTTGTGTCATGCACAAA GTAGATGTCC-30 ** Spl-P2 (linker-specific primer): 50 -TCGTACGAGAATCGCTGTCC TCTCC-30 PCR condition: same as the 1st PCR. 9. Check for PCR product by running 5 ll of each sample in a 2% agarose gel containing ethidium bromide for visualization. 10. Run remaining volume of PCR product (30–40 ll) in a 2% agarose gel containing ethidium bromide for sample preparation. 11. Gel extraction of bands under UV and proceed to purification using Qiagen QIAqucik gel extraction kit (using manufacturer’s instructions) – resuspend in either 25 or 50 ll DW, depending on the initial intensity of the PCR-band. 12. Proceed to sequencing using standard Sanger sequencing techniques. Usually, 1 ll of the purified PCR-band product is adequate. Transposon-specific 2nd PCR primer (T/BAL) was used as the sequencing primer. 2.5. The identification of mutant genes by 30 RACE (Fig. 3) 2.5.1. Isolation of RNA We routinely use tail clips or ear biopsy from rats (‘‘green rat” in gene-trapped event in Fig. 1c) as a source of RNA isolation. Here we describe a protocol using TRIzol (Invitrogen). 1. Add 1 ml of TRIzol to tail clips (2–3 mm) or ear biopsy (approximately 50 mg) and homogenize. RNA of animal tissues are easily degraded if not homogenized shortly after dissection. Since it is difficult to conduct simultaneous sampling and homogenization at the animal facility, samples are placed in RNAlater (Ambion) and stored at room temperature during the clipping procedure. It is important to remove RNAlater from the clippings before adding TRIzol. We remove all traces of RNAlater by placing clippings briefly on paper towel. 2. Incubate homogenized samples for 5 min at room temperature to allow for complete dissociation of nucleoprotein complexes. 3. Add 0.2 ml of chloroform for each ml of TRIzol reagent used in step (1). 4. Shake tubes vigorously by hands for 15 s and allow samples to sit at room temperature for 2–3 min. 5. Centrifuge at 15,000 rpm for 5 min at 4 °C. 6. Transfer aqueous phase to a new tube. 7. Add 0.5 ml of isopropyl alcohol, mix well, and incubate samples at room temperature for 10 min. 8. Centrifuge at 15,000 rpm for 10 min at 4 °C. 9. Remove the supernatant and wash RNA pellet once with RNase-free 75% ethanol. 10. Centrifuge at 15,000 rpm for 5 min at 4 °C. 11. Remove the supernatant and briefly dry RNA pellet. Avoid complete drying because solubility of RNA will decrease. Dissolve RNA pellet in RNase-free water. 12. Take OD to measure RNA concentration and store at 70 °C until required. 2.5.2. Reverse transcription Following is the protocol for first strand cDNA synthesis using Superscript II reverse transcriptase (Invitrogen). 1. Set up following mixture.
8-2. 2nd PCR reaction Set up following PCR reaction: 1st PCR product Buffer (10) dNTP (10 mM) T/BAL* (10 lM) Spl-P2** (10 lM) HotStarTaq DW Total
*
1 ll 5 ll 1 ll 1 ll 1 ll 0.25 ll 40.75 ll 50 ll
Total RNA Oligo-dT primer* (20 lM) dNTP (10 mM) RNase-free water Total *
1 ll (50 ng 2 lg) 1 ll 1 ll 10 ll 13 ll
Oligo-dT primer sequence: 50 -GGAGCAAGCAGTGGTAACAACGCA GAGTACCGATCAGTTGCTCTGGTGTCCGTGTCCTACTTTTTTTTTTTTTTT
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TTTTTTTTTTTTTTTVN-30 . V indicates degenerate sequence of A, G or C, whereas N denotes degenerate sequence of A, G, T, or C. 2. Incubate at 70 °C for 5 min, and chill on ice. 3. Spin down and add the following mixture. 4 ll 2 ll 1 ll 7 ll
RT buffer (5) DTT (0.1 M) RNasin (Promega) Total
4. Incubate at 42 °C for 2 min 5. Add 1 ll of Superscript II. It is desirable to set up another reaction as a negative control containing water instead of the Superscript II. 6. Incubate at 42 °C for 50 min 7. Heat-inactivate Superscript II at 70 °C for 15 min 2.5.3. Nested PCR 1. 1st PCR reaction Expand high-fidelity PCR system (Roche Diagnostics) was used to accommodate amplification of long transcripts. 1 ll 5 ll 1 ll 2.5 ll 2.5 ll 0.75 ll 37.25 ll 50 ll
cDNA Buffer* (10) dNTP (10 mM) EGFP-4U** (10 lM) RC1*** (10 lM) Polymerase Water Total
*
Several buffer components are supplied in the kit. Buffer 2 was used as a first choice. ** EGFP-4U (transposon-specific primer): 50 -CCCTGAGCAAAGA CCCCAACGAGAAGC-30 . *** RC1 (Oligo-dT-specific primer): 50 -GGAGCAAGCAGTGGTAACA ACGCAGAGTAC-30 . PCR condition:
*
EGFP-5U (transposon-specific primer): 50 -GCGATCACATGGTCCT GCTGGAGTTCGTG-30 . ** RC2 (Oligo-dT-specific primer): 50 -CGATCAGTTGCTCTGGTGT CCGTGTCCTAC-30 . PCR condition: same as the 1st PCR. 3. Check for PCR product by running 5 ll of the PCR product in a 1–2% agarose gel. 4. Run the remaining volume of PCR reaction product (30–40 ll) in agarose gel. 5. Gel extraction of bands under UV and proceed to purification using Qiagen QIAquick gel extraction kit (using manufacture’s instructions) – resuspend in either 20 or 50 ll DW, depending on the initial intensity of the PCR-band. 6. Proceed to sequencing using standard Sanger sequencing techniques. 3. Concluding remarks Despite its importance as an animal model for human diseases, generation of mutant rat resource lags far behind the mutant resources currently available for the mouse model. This is due to the lack an efficient high throughput mutagenesis platform. The simplicity of the transposon-based mutagenesis approach described in the current paper will help open and expand an area of research previously unavailable for the rat animal model and hopefully establish a mutant rat resource that will greatly benefit the biomedical research. References
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
94 °C 94 °C 60 °C 68 °C 68 °C 25 °C
5 min 1 min 1 min 1 min 5 min Hold
1 cycle 20 cycle 1 cycle
[19] [20] [21]
2. 2nd PCR 1st PCR product Buffer (10) dNTP (10 mM) EGFP-5U* (10 lM) RC2** (10 lM) Polymerase Water Total
[14] [15] [16] [17] [18]
[22] [23]
1 ll 5 ll 1 ll 2.5 ll 2.5 ll 0.75 ll 37.25 ll 50 ll
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Methods journal homepage: www.elsevier.com/locate/ymeth
Generating mutant rats using the Sleeping Beauty transposon system Kazuhiro Kitada a, Vincent W. Keng b, Junji Takeda c,d, Kyoji Horie c,* a
Laboratory of Mammalian Genetics, Division of Genome Dynamics, Creative Research Initiative ‘‘Sousei”, Hokkaido University, North 10 West 8, Kita-ku, Sapporo 060-0810, Japan Masonic Cancer Center and Center for Genome Engineering, University of Minnesota, 420 Washington Avenue SE, Minneapolis, MN 55455, USA Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan d Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan b c
a r t i c l e
i n f o
Article history: Accepted 10 April 2009 Available online 3 May 2009 Keywords: Mutagenesis Transposon Sleeping Beauty Rat
a b s t r a c t The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 30 -rapid amplification of cDNA ends (30 RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The laboratory rat (Rattus norvegicus) remains the major animal model system inside the pharmaceutical and biomedical research industries largely because ‘‘size does matter”. Importantly, rat models tend to be disease based, generating more information about the actual disease of interest, rather than just being an animal model. Unfortunately, geneticists have long preferred the mouse model because of its smaller size, which simplified housing requirements, and the availability of many coat color and mutants exhibiting Mendelian patterns of inheritance. In addition, the availability of mouse embryonic stem (ES) cells for gene-specific characterization and knockout studies has made this model indispensable. Although the availability of rat transgenic technologies have allowed advances in the field of gene-specific analysis, the lack of rat ES cells has hampered any further progress in this field. Germline-competent rat ES cells has been reported recently and may revolutionize this field [1,2]. However, the robustness for large-scale generation of mutant rats remains to be tested. Chemical mutagenesis has been widely used in the rat [3], however, a large portion of induced mutations are point-mutation and thus not null alleles in most cases, and moreover, identifying the causative gene seems to be a challenging task by this method. * Corresponding author. Fax: +81 6 6879 3266. E-mail address: [email protected] (K. Horie). 1046-2023/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2009.04.010
Sleeping Beauty (SB) transposon system is a novel genetic tool developed around a decade ago [4]. It consists of two components: The SB transposon, which is excised and reinserted into other locations of the genome, and the SB transposase catalyzes this reaction. Since most of the vertebrate transposase genes are inactivated during evolution, the SB transposase was reconstructed by extensive manipulation of inactive transposase copies from the fish genome. The SB transposon contains inverted repeat/direct repeat (IR/DR) elements at either ends (Fig. 1a), which are essential sequence for the recognition and mobilization by the SB transposase. Recently, others and we have demonstrated the effectiveness of SB for large-scale germline mutagenesis in mice [5–7]. Analysis of the SB transposition sites revealed preference of transposition on the donor-site chromosome, which varied between 60% to 80% amongst SB transposon-transgenic lines, whereas remaining 20– 40% was distributed in various locations of the genome [5,6]. Many of the transpositions on the donor-site chromosome were clustered within 3–4 Mb from the donor site [5,6]. Others and we further applied the SB transposon system in rats, and succeeded in rapid generation of mutants [8,9]. In the present paper, we describe a protocol for the generation and analysis of the mutant rats based on the SB transposon vectors developed in our laboratory [8]. It should be noted that various transposon systems have been developed recently and some of them have been shown to be active in mammalian cells, such as Tol2 [10], PiggyBac [11], Minos [12], and Frog Prince [13]. Although
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a
SB transposon
Transposon vector
IR/DR
IR/DR SA IRES LacZ pA
Promoter trap system for X-gal detection
Transposase vector
b
E1
Plasmid backbone
CAG GFP SD
PolyA trap system for noninvasive GFP detection
CAG SB pA
E2
E3 SA IRES LacZ pA (A)n
Truncated endogenous protein
c
SB
X
E4
CAG GFP SD (A)n
GFP
ß-gal
Gfp GFP
SB Transposase Tg rat
Transposon Transposon TgTgrat rat
SB GFP Gfp
X
WT
Double Tg rat "seed rat"
Wild-type rat
Screening for GFP-positive rats
Gfp GFP No gene-trapped event
Bright-field
Fluorescence
Gfp GFP Gene-trapped event
DNA
RNA
LM-PCR (Fig. 2)
3’ RACE (Fig. 3)
Production of progeny, Intercross for homozygous mutant generation, & phenotypic analysis
Fig. 1. Overview of the mutagenesis strategy. (a) Structure of vectors used to generate transgenic lines. Transgenic rats bearing the transposon vector (top) and the transposase vector (bottom) were generated independently, and mated to generate doubly-transgenic rats as shown in (c). IR/DR is the recognition sequence of the SB transposase. The region flanked by IR/DRs is excised by the SB transposase and inserted into other locations of the genome. SA, splice acceptor; SD, splice donor; IRES, internal ribosome entry site; pA, polyA signal; CAG, cytomegalovirus enhancer/chicken beta-actin chimeric promoter; IR/DR, inverted repeat/direct repeat. (b) Scheme of the promoter- and polyA-traps. In this example, the SB transposon vector is inserted into the 2nd intron. The transcript from the inserted gene is trapped by the splice acceptor of the transposon vector. As a result, production of the endogenous protein is disrupted and beta-galactosidase from the LacZ gene is expressed, which can be used as a reporter for the expression profile of the mutated gene in various tissues. The GFP reporter is driven by the constitutively active CAG promoter and is expressed as a result of the splicing between vector-derived splice donor and the splice acceptor of the downstream exon. E, exon. (c) Breeding scheme for the generation of mutant progeny and an example of the screening for mutants based on GFP expression. The rat at the top of the photograph is GFP-positive and therefore presumably an insertional mutant, while the rat at the bottom is GFP-negative and therefore, a non-mutant.
their application for insertional mutagenesis in rat remains to be addressed, it is conceivable that some of them will have different characteristics compared with SB, such as different patterns of transposition distribution [14] and different rates of transposition events. Therefore, it is highly likely that these DNA-type transposable elements will complement SB as a mutagenesis tool. Most of the protocol described in this paper can be easily modified and applicable to other transposon systems by simple modification.
2. Description of method 2.1. Overview (Fig. 1) Vector structures and general mutagenesis scheme are shown in Fig. 1. The initial part is to generate two transgenic rat lines, one containing a donor concatemer of transposon gene-trap vec-
tors and the other containing a source of transposase (Fig. 1a). The transposon gene-trap vector concatemer source consists of a promoter-trap component and a polyA-trap component. The promoter-trap component consists of human BCL2 intron 2/exon3 splice acceptor (SA) [15], encephalomyocarditis virus internal ribosome entry site (IRES), the LacZ gene and rabbit beta-globin polyA signal (pA). The polyA-trap component contains the cytomegalovirus (CMV) enhancer/chicken beta-actin chimeric promoter (CAG promoter) [16], the GFP gene, and splice donor (SD) from the mouse Hprt gene exon 8/intron 8 region which is followed by the mRNA instability signal derived from the human granulocyte-macrophage colony-stimulating factor cDNA [15]. The source of transposase is made using the CAG promoter driving SB transposase. These lines are mated with each other to generate doubly-transgenic rats, and the doubly-transgenic rats are bred to wild-type rats (Fig. 1c). Transposition in the germline of the doubly-transgenic rats (‘‘seed rat” in Fig. 1c) will generate heterozy-
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gous mutant rats in the progeny. If transposon integration occurs within a gene, the promoter-trap component allows for the detection of LacZ staining, relying on the endogenous promoter (Fig. 1b). In addition, the polyA-trap component relies on the polyA signal of the endogenous gene to produce the GFP protein and fluorescence can be detected non-invasively (Fig. 1b and c). Mutated genes are identified with either of the two methods: (1) Linker ligation-mediated PCR (LM-PCR) (Fig. 2), and (2) 30 -rapid amplification of cDNA ends (30 RACE) (Fig. 3). The former method determines the exact vector insertion site at the nucleotide level. The latter method amplifies trapped transposon-endogenous exon sequence from mRNA. These methods are complementary to each other in identifying the insertion site of the transposon vector. The first approach is relatively simple and straightforward but prediction of the mutated gene is dependent on the rat genome annotation database, in which the mutated gene may not be mapped yet. The latter method may reveal novel transcripts, which are not yet annotated in the genome database, but the transposon insertion site could be unexpectedly distant from the trapped site. 50 RACE is another useful approach to determine novel transcripts and has been widely used in gene trap in mouse embryonic stem cells [17]. We use 30 RACE because we routinely use ear or tail clip as a source of RNA and the mutated gene under study may not be expressed there.
Transposition site
Transposition may occur in somatic cells of mutant rats when the SB transposase is not segregated. Our experience indicates that its frequency is not high enough to create confusion in the analysis of germline transpositoin sites. However, it is advisable to select for mutant rats that do not contain the SB transposase in order to avoid continuous germline transposition in the progeny. 2.2. Generation of transgenic rats containing the transposon and the transposase vectors 2.2.1. Consideration for vector construction For generation of transgenic rats, plasmid backbone sequence is usually removed from vector DNA prior to pronuclear injection. We followed this rule for the injection of the SB transposase vector. In contrast, we did not remove the plasmid backbone (pBluescript and neo gene cassette) for the injection of the transposon vector (Fig. 1a). These sequences are not required for the function of the transposon. However, our experience suggests that the presence of this region induces methylation of the transposon DNA and inactivate the CAG promoter [18]. As a result, GFP fluorescence from the donor site is prevented. In contrast, the CAG promoter is activated at the transposition site because the transposon is not flanked by the plasmid backbone any more. As a result, gene insertion events can be
Vector concatemer at the donor site
Transposon
Plasmid backbone Genomic sequence
X
IR/DR
X
X
IR/DR
IR/DR
Y
X
IR/DR
Digestion with enzyme “X” X
X
Y
X
X
Linker ligation Y
Linker
Digestion with enzyme “Y” Y
Y
1st PCR primers
1st PCR No amplification 2nd PCR primers 2nd PCR
Sequencing
A
G
T
C
Fig. 2. Linker ligation-mediated PCR (LM-PCR). Amplification of rat genomic sequence at transposition sites (left) and the elimination of undesired amplification products derived from the transposon vector donor concatemer (right). Genomic DNAs from GFP-positive rats (Fig. 1c) are digested with the enzyme ‘‘X”, a linker DNA is ligated, and transposition sites are amplified by nested-PCR using vector-specific primers and linker-specific primers. To avoid amplification of the vector concatemer at the donor site, linker-ligated DNAs are digested with the enzyme ‘‘Y” which is located just outside of the IR/DR. We usually use 4-base cutters for enzyme ‘‘X” and 6-base cutters for enzyme ‘‘Y”.
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SA IRES LacZ pA
CAG GFP SD AAAA------AA
GFP
Trapped sequence AAAA------AA
mRNA
DNA from step (1) Buffer (5) dNTP (10 mM) MgCl2 (50 mM) Primer mix* (10 lM each) Tfi DNA polymerase (Invitrogen) DW Total
1 ll 5 ll 0.5 ll 0.75 ll 0.5 ll 0.5 ll 16.75 ll 25 ll
Reverse transcription
mRNA
AAAA------AA
cDNA
TTTT----
Oligo-dT primer
* PCR primer pairs: 50 -AAGACCCATTTGCGACCAAGCTTTA-30 and 50 CCGAAGAACACCATCCCAACCGTGA-30 for SB transposase, and 50 -AA CGTCTATATCATGGCCGACAA-30 and 50 -TGGGGGTGTTCTGCTGGTAG TGGTC-30 for SB transposon lines, respectively. 3. Perform PCR reaction. PCR condition:
1st PCR 1st PCR primer TTTT----
1st PCR primer AAAA---TTTT---AAAA---TTTT---AAAA---TTTT---2nd PCR primers 2nd PCR
AAAA---TTTT---AAAA---TTTT---AAAA---TTTT----
Sequencing
A
G
T
C
Fig. 3. 30 -rapid amplification of cDNA ends (30 RACE). Total RNA from GFP-positive rats (Fig. 1a) are reverse transcribed with the oligo-dT primer, and trapped sequences are amplified by nested-PCR using GFP-specific primers and oligo-dTspecific primers. Abbreviations are as described in Fig. 1.
visualized as ‘‘green rats” (Fig. 1c). Furthermore, our in vitro study indicated that the DNA methylation and heterochromatinization of the transposon donor site significantly enhances SB transposition [19,20]. It should be noted that the enhancement of transposition is not necessarily seen in other transposon systems [14]. 2.2.2. Maintenance of transgenic rat lines It is important that transgenic lines harboring SB transposase and transposon vector should be maintained independently. Maintenance as a doubly transgenic line is not recommended because the structure and copy number of the donor concatemer may be altered due to germline transposition of the SB transposon. The PCR genotyping protocol of the SB transposase and the SB transposon lines that were reported in our study [8]. 1. Dilute genomic DNA to a 100 ng/ll concentration using distilled water (DW). 2. Set up the following PCR reaction:
94 °C 94 °C 55 °C 72 °C 72 °C 25 °C
2 min 30 s 1 min 45 s 10 min Hold
1 cycle 30 cycle 1 cycle
4. Separate PCR products in a 2–4% agarose gel containing ethidium bromide for visualization. PCR amplicon of 191-bp will be seen in transgenic lines harboring the SB transposase, and a 118-bp PCR product will be seen in transgenic lines harboring the SB transposon vector, respectively. 2.2.3. Selection for GFP-negative rat As mentioned above, it is expected that a plasmid backbone of the transposon vector should prevent GFP expression due to the highly methylated state of the transposon donor concatemer. However, some transposon vector-carrying transgenic lines do reveal detectable GFP expression even though no transposase-induced transposition had occurred. Such GFP-positive lines must be excluded from further analyses. We recommend that this step should be performed at G1 generation, and not at the founder generation, considering possible mosaicism is occasionally seen in the founder generation. Selection for GFP-negative rat can be performed as described in the Section 2.3.1. 2.2.4. Testing for the effect of homozygosity at the donor site It is reported that ‘‘local hopping” events are frequently observed for the SB transposon system [5–7]. Segregation between a transposed site and the transposon vector donor concatemer site may not be easily achieved by recombination in some cases. In order to precisely evaluate trapped gene events, it is essential to confirm that homozygosity of the donor concatemer site does not generate any overt phenotype before utilizing the transgenic line for further analyses. For this purpose, G2 offspring are generated by intercrossing G1 transgene-positive rats described earlier in Section 2.2.2. Identification of homozygotes in the G2 offspring can be achieved by FISH (fluorescence in situ hybridization) analyses or the classical testcross analyses [21]. Selected G2 homozygotes are then aged and examined carefully. 2.2.5. Screening of the SB transgenic lines The expression level of the SB transposase varies among transgenic lines and some lines may not exhibit any expression. Therefore, it is recommended to analyze the expression of the SB transposase in the male germline and identify appropriate transgenic lines. The standard methods such as immunohistochemistry,
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northern blotting or RT-PCR can be used. Antibody against SB transposase is commercially available from R&D Systems. 2.3. Generation and identification of mutant rats Transgenic lines harboring SB transposase and transposon vector are intercrossed and doubly-transgenic rats are selected as described in Section 2.2.2. Doubly-transgenic rats ‘‘seed rat” are then mated with wild-type rats. Transposition in the germline of these doubly-transgenic rats will generate heterozygous mutant rats in the progeny. Mutant rats are easily and rapidly selected by a non-invasive method using a fluorescent microscope (Fig. 1c). We usually use male doubly-transgenic rats because more breeding pairs can be set up compared with female ‘‘seed rats”. 2.3.1. Detection of GFP-positive mutant rats using fluorescent microscope As GFP signals are generally weak in our gene-trapped mutant rats, the use of a fluorescent microscope with high enough sensitivity is essential and a hand-held UV light source is not suitable for this purpose. We routinely use a SMZ800 fluorescent dissecting scope (Nikon) with a filter block P-FLGFP-B (460–500 nm band pass excitor filter and 510–560 nm barrier filter). With the detection of a weak GFP signal, it is recommended that screening be performed on neonates around 3-days of age. Delayed screening should be avoided as hair development occurs and the detection of GFP signal can become very difficult. 2.3.2. Frequency of mutation in progeny In our transposon system, approximately 11% of newborn rats from doubly-transgenic rats are GFP-positive, even though this frequency seems to vary amongst transposon vector lines. In our hands, it is determined that the transposition events in germ line cells occur with a rate of approximately one transposition event per spermatozoon. 2.3.3. Consideration for possible chromosomal rearrangement during transposition It should be noted that chromosomal rearrangement near the donor concatemer might arise during the transposition reaction [22]. Since the SB transposon has a preference to transpose near the donor site [5–7], there is a possibility that some of the transposon insertions are tightly linked to the rearranged region and cannot be segregated easily. This possibility should be kept in mind during phenotypic analyses because the observed phenotypes could be due to the chromosomal rearrangement and not the transposition sites. Therefore, mapping the location of the insertion mutations is a critical part of their ultimate usefulness and should be evaluated in the initial phase of the phenotypic analysis. 2.4. The mapping of insertion sites by LM-PCR (Fig. 2) 2.4.1. Isolation of genomic DNA Tail or ear clips from rats (‘‘green rat” in gene-trapped event in Fig. 1c) can be used as a source of genomic DNA. The protocol described below is based on the classical method involving phenol/ chloroform extraction and ethanol precipitation techniques. We have also used column-based commercial kits (e.g. DNeasy Blood & Tissue Kit, Qiagen) and insertion sites could also be determined seamlessly. Since amplification by the LM-PCR protocol is robust, most DNA isolation methods that provide DNA quality compatible with Southern analysis will be adequate for LM-PCR. 1. Lyse source material (e.g. 0.5 cm of tail clip) in 1.5 ml microtubes using a DNA extraction buffer containing proteinase K (500 ll of DNA extraction buffer [10 mM Tris–
2.
3.
4.
5. 6. 7. 8. 9. 10. 11.
HCl, 1 mM EDTA, 1 SSC, 1% SDS] with 10 ll of proteinase K [10 mg/ml stock, Merck] added) – incubate at 55 °C overnight with shaking. Centrifuge at 15,000 rpm for 5 min at 4 °C to pellet undissolved material – transfer liquid phase to a fresh microtube using pipette tips cut at the end to avoid genomic DNA shearing. Add equal volume (500 ll) of phenol:chloroform (1:1 ratio) – mix for a few min by gentle inversion (this step may be repeated if protein interface contamination occurs). Centrifuge at 15,000 rpm for 10 min at 4 °C – transfer aqueous phase to a fresh microtube using pipette tips cut at the end to avoid genomic DNA shearing. Add 0.7 volume (350 ll) of isopropanol– mix for a few min by gentle inversion. Centrifuge at 15,000 rpm for 10 min at 4 °C to pellet genomic DNA precipitate. Discard supernatant – wash with 500 ll 70% ethanol. Centrifuge at 15,000 rpm for 10 min at 4 °C to pellet genomic DNA precipitate. Discard supernatant – dissolve genomic DNA in 50–200 ll TE (Tris–HCl 10 mM; EDTA 1 mM). Leave overnight at 4 °C to fully dissolve genomic DNA in TE. Mix gently, spin down and obtain OD reading at 260/280 nm to determine concentration and purity (260/280 ratio should be greater than 1.8).
2.4.2. Linker-ligation and PCR 1. Dilute genomic DNA to a 10 ng/ll concentration using DW. 2. Digest 100 ng of diluted genomic DNA at 37 °C for 2–3 h in a final volume of 50 ll using an appropriate 4-base restriction enzyme (enzyme ‘‘X” in Fig. 2). It should not cleave between the transposon-specific primer (see below, indicated by arrowhead in Fig. 2) and the end of the IR/DR. In the case of our SB transposon vector, the following enzymes (New England Biolabs) can be used: i. AluI, ii. MboI, iii. HaeIII 3. Heat-inactivate restriction enzymes by incubating at the following temperatures for 20 min: i. AluI, MboI – 65 °C, ii. HaeIII – 80 °C. 4. Linkers (Splinkerettes [23]) are prepared by annealing the following oligonucleotide sequences together (10 lM each): Spl-top 50 -CGAATCGTAACCGTTCGTACGAGAATTCGTACGAGAATCGCTGTCC TCTCCAACGAGCCAAGG-30 with either SplB-blunt 50 -CCTTGGCTCG TTTTTTTTTGCAAAAA-30 to obtain the final Spl-blunt linker; or Spl-top with SplB-sau 50 -GATCCCTTGGCTCGTTTTTTTTTGCAAAAA30 to obtain the Spl-sau linker with 50 -protruding end. Combination of the cleavage end and the linker is as follows: i. AluI, HaeIII– use Spl-blunt, ii. MboI – use Spl-sau. Set up following ligation reaction (Takara DNA ligation kit ver. 1) and incubate at 16 °C for at least 3 h: Enzyme-digested genomic DNA Appropriate linker Buffer A Buffer B Total
2 ll 1 ll 12 ll 3 ll 18 ll
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5. Purify using Qiagen QIAquick PCR purification kit (using manufacturer’s instructions) – resuspend in 40 ll DW. 6. Digest each sample with a 6-base restriction enzyme (or any appropriate rare cutter enzyme, indicated by enzyme ‘‘Y” in Fig. 2) that cleaves the transposon vector backbone sequence just outside of the IR/DR but does not cleaves between the transposon-specific primer (see below, indicated by arrowhead in Fig. 2) and the end of the IR/DR. This step prevents amplification of the transposon vector donor concatemer site. In our SB transposon vector, KpnI is appropriate for this purpose. In case the donor site is segregated during the breeding process, this step can be omitted. Set up the following reaction and incubate at 37 °C overnight to accomplish complete digestion. 39 ll 5 ll 5 ll 1 ll 50 ll
Linker-ligated/purified DNA Appropriate buffer (10) BSA (10) Enzyme (in excess) Total
7. Purify using Qiagen QIAqucik PCR purification kit (using manufacturer’s instructions) – resuspend in 50 ll DW (may omit if sample quantity is large and can proceed directly to nested-PCR). 8. Nested-PCR. 8-1. 1st PCR reaction Set up following PCR reaction: 1 ll 5 ll 1 ll 1 ll 1 ll 0.25 ll 40.75 ll 50 ll
DNA from step (7) Buffer (10) dNTP (10 mM) T/DR2* (10 lM) Spl-P1** (10 lM) HotStarTaq (Qiagen) DW Total
* T/DR2 (transposon-specific primer): 50 -CTGGAATTGTGATACA GTGAATTATAAGTG-30 . ** Spl-P1 (linker-specific primer): 50 -CGAATCGTAACCGTTCGTAC GAGAA-30 . Transposon-specific primer is designed in the IR/DR region downstream of the polyA-trap component. Therefore, genomic sequence at the downstream of the transposon insertion site is amplified. PCR condition:
95 °C 95 °C 55 °C 72 °C 72 °C 25 °C
15 min 1 min 1 min 1 min 7 min Hold
1 cycle 30 cycle 1 cycle
T/BAL (transposon-specific primer): 50 -CTTGTGTCATGCACAAA GTAGATGTCC-30 ** Spl-P2 (linker-specific primer): 50 -TCGTACGAGAATCGCTGTCC TCTCC-30 PCR condition: same as the 1st PCR. 9. Check for PCR product by running 5 ll of each sample in a 2% agarose gel containing ethidium bromide for visualization. 10. Run remaining volume of PCR product (30–40 ll) in a 2% agarose gel containing ethidium bromide for sample preparation. 11. Gel extraction of bands under UV and proceed to purification using Qiagen QIAqucik gel extraction kit (using manufacturer’s instructions) – resuspend in either 25 or 50 ll DW, depending on the initial intensity of the PCR-band. 12. Proceed to sequencing using standard Sanger sequencing techniques. Usually, 1 ll of the purified PCR-band product is adequate. Transposon-specific 2nd PCR primer (T/BAL) was used as the sequencing primer. 2.5. The identification of mutant genes by 30 RACE (Fig. 3) 2.5.1. Isolation of RNA We routinely use tail clips or ear biopsy from rats (‘‘green rat” in gene-trapped event in Fig. 1c) as a source of RNA isolation. Here we describe a protocol using TRIzol (Invitrogen). 1. Add 1 ml of TRIzol to tail clips (2–3 mm) or ear biopsy (approximately 50 mg) and homogenize. RNA of animal tissues are easily degraded if not homogenized shortly after dissection. Since it is difficult to conduct simultaneous sampling and homogenization at the animal facility, samples are placed in RNAlater (Ambion) and stored at room temperature during the clipping procedure. It is important to remove RNAlater from the clippings before adding TRIzol. We remove all traces of RNAlater by placing clippings briefly on paper towel. 2. Incubate homogenized samples for 5 min at room temperature to allow for complete dissociation of nucleoprotein complexes. 3. Add 0.2 ml of chloroform for each ml of TRIzol reagent used in step (1). 4. Shake tubes vigorously by hands for 15 s and allow samples to sit at room temperature for 2–3 min. 5. Centrifuge at 15,000 rpm for 5 min at 4 °C. 6. Transfer aqueous phase to a new tube. 7. Add 0.5 ml of isopropyl alcohol, mix well, and incubate samples at room temperature for 10 min. 8. Centrifuge at 15,000 rpm for 10 min at 4 °C. 9. Remove the supernatant and wash RNA pellet once with RNase-free 75% ethanol. 10. Centrifuge at 15,000 rpm for 5 min at 4 °C. 11. Remove the supernatant and briefly dry RNA pellet. Avoid complete drying because solubility of RNA will decrease. Dissolve RNA pellet in RNase-free water. 12. Take OD to measure RNA concentration and store at 70 °C until required. 2.5.2. Reverse transcription Following is the protocol for first strand cDNA synthesis using Superscript II reverse transcriptase (Invitrogen). 1. Set up following mixture.
8-2. 2nd PCR reaction Set up following PCR reaction: 1st PCR product Buffer (10) dNTP (10 mM) T/BAL* (10 lM) Spl-P2** (10 lM) HotStarTaq DW Total
*
1 ll 5 ll 1 ll 1 ll 1 ll 0.25 ll 40.75 ll 50 ll
Total RNA Oligo-dT primer* (20 lM) dNTP (10 mM) RNase-free water Total *
1 ll (50 ng 2 lg) 1 ll 1 ll 10 ll 13 ll
Oligo-dT primer sequence: 50 -GGAGCAAGCAGTGGTAACAACGCA GAGTACCGATCAGTTGCTCTGGTGTCCGTGTCCTACTTTTTTTTTTTTTTT
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TTTTTTTTTTTTTTTVN-30 . V indicates degenerate sequence of A, G or C, whereas N denotes degenerate sequence of A, G, T, or C. 2. Incubate at 70 °C for 5 min, and chill on ice. 3. Spin down and add the following mixture. 4 ll 2 ll 1 ll 7 ll
RT buffer (5) DTT (0.1 M) RNasin (Promega) Total
4. Incubate at 42 °C for 2 min 5. Add 1 ll of Superscript II. It is desirable to set up another reaction as a negative control containing water instead of the Superscript II. 6. Incubate at 42 °C for 50 min 7. Heat-inactivate Superscript II at 70 °C for 15 min 2.5.3. Nested PCR 1. 1st PCR reaction Expand high-fidelity PCR system (Roche Diagnostics) was used to accommodate amplification of long transcripts. 1 ll 5 ll 1 ll 2.5 ll 2.5 ll 0.75 ll 37.25 ll 50 ll
cDNA Buffer* (10) dNTP (10 mM) EGFP-4U** (10 lM) RC1*** (10 lM) Polymerase Water Total
*
Several buffer components are supplied in the kit. Buffer 2 was used as a first choice. ** EGFP-4U (transposon-specific primer): 50 -CCCTGAGCAAAGA CCCCAACGAGAAGC-30 . *** RC1 (Oligo-dT-specific primer): 50 -GGAGCAAGCAGTGGTAACA ACGCAGAGTAC-30 . PCR condition:
*
EGFP-5U (transposon-specific primer): 50 -GCGATCACATGGTCCT GCTGGAGTTCGTG-30 . ** RC2 (Oligo-dT-specific primer): 50 -CGATCAGTTGCTCTGGTGT CCGTGTCCTAC-30 . PCR condition: same as the 1st PCR. 3. Check for PCR product by running 5 ll of the PCR product in a 1–2% agarose gel. 4. Run the remaining volume of PCR reaction product (30–40 ll) in agarose gel. 5. Gel extraction of bands under UV and proceed to purification using Qiagen QIAquick gel extraction kit (using manufacture’s instructions) – resuspend in either 20 or 50 ll DW, depending on the initial intensity of the PCR-band. 6. Proceed to sequencing using standard Sanger sequencing techniques. 3. Concluding remarks Despite its importance as an animal model for human diseases, generation of mutant rat resource lags far behind the mutant resources currently available for the mouse model. This is due to the lack an efficient high throughput mutagenesis platform. The simplicity of the transposon-based mutagenesis approach described in the current paper will help open and expand an area of research previously unavailable for the rat animal model and hopefully establish a mutant rat resource that will greatly benefit the biomedical research. References
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
94 °C 94 °C 60 °C 68 °C 68 °C 25 °C
5 min 1 min 1 min 1 min 5 min Hold
1 cycle 20 cycle 1 cycle
[19] [20] [21]
2. 2nd PCR 1st PCR product Buffer (10) dNTP (10 mM) EGFP-5U* (10 lM) RC2** (10 lM) Polymerase Water Total
[14] [15] [16] [17] [18]
[22] [23]
1 ll 5 ll 1 ll 2.5 ll 2.5 ll 0.75 ll 37.25 ll 50 ll
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