Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method
© Kage-Nakadai et al; licensee BioMed Central Ltd. 2012
Received: 22 October 2011
Accepted: 5 January 2012
Published: 5 January 2012
Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations.
We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans.
Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.
The development of methods to introduce exogenous DNA into animals has allowed for diverse genetic manipulations in many organisms. In Caenorhabditis elegans, transgenic strains are typically generated by injecting DNA into the syncytial germ cells of the hermaphrodite gonad to form multi-copy extrachromosomal arrays . These transgenes are semi-stable; transgenic animals are mosaic in that some cells lose the extrachromosomal array, and transmission of arrays to the next generation is partial . Extrachromosomal arrays contain hundreds of copies of the injected DNA , leading to silencing of the transgene expression in the germline . Although extrachromosomal arrays can be integrated into the chromosomes by gamma-ray irradiation or ultraviolet (UV) [4, 5], integrated arrays still contain a high copy-number of transgenes that seldom escape gene silencing.
Methods using microparticle bombardment were developed to create low-copy chromosomal integrated lines . The biolistic technique allows for direct integration of small amounts of exogenous DNA into the chromosomes, avoiding the formation of extrachromosomal arrays. Not every bombardment, however, produces integrant animals because of the low frequency of events, although large number of animals can be bombarded at once (~104/bombardment) . More recently, techniques using Mos1 transposons were developed and are frequently used to generate single-copy gene insertions . This technique, called Mos1-mediated single-copy insertion (MosSCI), is based on homologous recombination: A double-stranded break in the chromosome mediated by Mos1 excision is repaired with an exogenously supplied template carrying the gene of interest and homology arms, generating the designed single copy insertion . MosSCI methods, in which a recipient strain carrying a Mos1 element is microinjected with a targeting vector and a Mos1 transposase expression vector, exhibit a high frequency of insertion, and injection of 20 worms is enough to obtain integrant animals . In the MosSCI method, large sized targeting vectors that contain positive selection marker, 5'- and 3'- homology arms, and gene of interest must be constructed for each insertion, although a Gateway-compatible tool kit for MosSCI has been developed .
In reverse genetic studies, ultraviolet trimethylpsoralen (UV/TMP), which induces a small deletion in the chromosomes, has been widely used to generate deletion mutants [9–11]. In addition to deletions, insertions of unexpected DNA fragments are often observed, suggesting that non-homologous DNA is used as a template in end-joining repair mechanisms. UV irradiation (wavelength 365 nm) and TMP treatment has a higher mutation frequency and less rearrangement of chromosomes, such as inversion and translocation, compared to UV irradiation (wavelength 254 nm), which is used for insertion of extrachromosomal arrays [5, 11, 12].
In the present study, we developed a technique using UV/TMP that produces single- or low- copy gene integrations from extrachromosomal arrays. In this method, we used a positive selection marker flanked by LoxP sites, allowing the marker to be excised by the Cre recombinase.
Results and discussion
Transgene integration using UV/TMP methods
Our methods were based on random integrations of transgenes into the chromosomes from multi-copy extrachromosomal arrays. We adopted a new positive-negative selection strategy as follows: Positive selection was based on rescue of the vps-45 mutant phenotype. vps-45 mutants are unable to grow and reproduce normally at 20°C . As a result, only vps-45 mutants carrying the positive selection marker, the vps-45 mini gene, grow and reproduce, allowing for easy identification of the transformants. To discriminate single- or low-copy integrations from extrachromosomal arrays and multi-copy integrations, we used ben-1 as a negative selection marker. Mutants of ben-1, which encodes a ß-tubulin of C. elegans, are resistant to an anti-tubulin drug benzimidazole: mutants grow paradoxically more quickly than wild-type animals, which are severely unhealthy, dumpy, and uncoordinated in movement on benzimidazole-containing selection media . Thus, ben-1 mutants not carrying the negative selection marker, the ben-1 gene, predominantly grow and reproduce on the selection media, enabling differentiation of low copy integrants from Ex arrays and multi-copy integrants that are highly likely to have the ben-1 gene (Additional file 1, Figure S1).
Frequency of integrations
Frequency of integrations using UV/TMP methods
(Is lines/P0 animals)
Germline expression of transgenes
Copy number of integrated transgenes
To determine the copy number of insertions, we performed quantitative PCR using purified genome DNA as a template. We designed primer sets located within an exon of the vps-45 gene to detect both endogenous vps-45 and exogenous vps-45 mini gene insertions. Eight HBG integrant strains were tested and compared to wild-type N2, which has two copies of the vps-45 gene. As a result, five strains in the tm234(ben-1);tm246(vps-45)-background: tmIs840, tmIs841, tmIs843, tmIs844, and tmIs864 showed almost the same relative amount as N2, suggesting that these strains had two copies of the vps-45 gene, probably because they were homozygous for alleles carrying a single insertion site (Figure 1B). On the other hand, two strains in the tm234(ben-1);tm246(vps-45)-background: tmIs839, tmIs842 showed two times higher amounts, and one strain in the tm234(ben-1);tm246(vps-45)-background: tmIs852 showed three or four times higher amounts compared to N2 (Figure 1B), suggesting that these strains contain a low-copy, but not a single-copy number, of transgenes. Examination of tm234;tm246;tmEx2274, the parent strain, revealed that the parent Ex strain contained hundreds of copies of transgenes (Figure 1B), consistent with previous studies . These results strongly suggest that UV/TMP methods produce single- or low-copy integrations. We also determined the copy number of the vps-45 gene in four HSP integrated strains, revealing the single copy insertion of the vps-45 mini gene in tmIs893 and tmIs895 and low-copy, but not single-copy insertion, in tmIs892 and tmIs894 (Figure 2C, left). We also examined the copy number of P hsp-16.1 ::venus using primers that amplify the hsp-16.1 promoter region, and found that tmIs892, tmIs893, tmIs895 contained a single-copy insertion of P hsp-16.1 ::venus (Figure 2C, right). These results demonstrate that the UV/TMP method is useful for low-copy insertion of genes of interest and also highly likely to produce single-copy insertion.
Genomic-excision using Cre mRNA microinjection
Mapping of Cre-excised insertion sites
In the present study, we developed an alternative method to promote low copy/single copy integration using UV/TMP. This method is based on the familiar extrachromosomal transgenics and does not require any special equipment, and should be very easy for most C. elegans researchers. Our methods exhibited higher efficiency when compared to the microparticle bombardment system, and lower efficiency when compared to MosSCI (Additional file 2, Table S1). The low-copy of integrated transgenic lines rather than single-copy lines, however, may be precious in some cases, e.g. when proteins of interest may not be sufficiently expressed by single-copy transgenes. Indeed, a few-copies of P hsp-16.1 ::venus transgene exhibited brighter fluorescence and expressed several times higher Venus mRNA than the single-copy transgene (data not shown and Additional file 3, Figure S2). We adopted vps-45 as a positive selection marker and ben-1 as a negative selection marker, whereas previous microparticle bombardment and MosSCI systems used other selection markers (Additional file 2, Table S1). The vps-45 mutants showed a distinct ts phenotype and ben-1 mutants exhibited a strong benzimidazole-resistant phenotype, both of which were fully rescued by transgenes, enabling desired integrants to be easily identified. Our selection strategy offers a new gene set for positive-negative selection in many other screens. We used a floxed positive selection marker to be excised afterwards. Previous studies showed that Cre-mediated LoxP excision successfully occurs in the extrachromosomal arrays [15, 16]. In these cases, however, excision was limited in a small portion of hundreds of floxed transgenes. Our results demonstrate Cre-mediated chromosomal and complete excision in C. elegans for the first time. The UV/TMP integration method provides a useful approach to generating single/low-copy gene integrations.
C. elegans strains were cultured using standard techniques . The wild-type strain Bristol N2 was obtained from the Caenorhabditis Genetics Center. Strains carrying the following mutations were obtained from the UV/TMP mutagenized library, as described previously  and identified by PCR amplification with primers spanning the deletion region of ben-1(tm234)III and vps-45(tm246)X, as described previously [11, 13]. The mutants were backcrossed four times with N2. Primers used for PCR genotyping were as follows: tm234_1stround, 5'-ACGTGGGAATGGAACCATGT-3', 5'-TCTCCATTTCCTCTTCCTCC-3'; tm234_2ndround, 5'-CTCCGGACATTGTAACGGAA-3', 5'-CCCTCCATTTGAAAGAGTCC-3'; tm246, 5'-CGCAATTGGATACTACTTGT-3', 5'-TCTCCTGCTCTACTTCTGCT-3'.
Constructs and transgenic lines
The positive selection marker plasmid (pFX_HBG_Lw_vps-45) was constructed by subcloning the HBG1 sequence (60 bp of partial human ß-globin sequence), wild-type LoxP sequence (ATAACTTCGTATAGCATACATTATACGAAGTTAT) , and vps-45 mini gene (eft-3p::vps-45cDNA::unc-86 3'-UTR), into pBluescriptII MCS. The whole sequence is available upon request. The negative selection marker plasmid (pGEMT_ben-1(+)) was constructed by TA-cloning in which the ben-1 genome (2414 bp of the 5' upstream region followed by the coding sequence and 785 bp of the 3'-UTR) was subcloned into the pGEMT-easy vector. To generate the P hsp-16.1 ::venus plasmid (pFX_hs::venusT), 117 bp of the upstream genomic region of the hsp-16.1 gene was cloned into the 5' region of the Venus expression vector in-frame . To generate tmEx2274 transgenic animals, pFX_HBG_Lw_vps-45, pGEMT_ben-1(+) (80 ng/μl) were co-injected with P myo-2 ::venus as an injection marker (20 ng/μl) into tm234(ben-1);tm246(vps-45). To generate tmEx2677 transgenic animals, pFX_HBG_Lw_vps-45, pGEMT_ben-1(+) and pFX_hs::venusT were co-injected at 67 ng/μl each along with P myo-2 ::venus as an injection marker (20 ng/ml) into tm234(ben-1);tm246(vps-45).
UV/TMP treatment and positive-negative selection
Treatment with UV and TMP was conducted as described below. TMP (Wako) was dissolved completely in acetone at a concentration of 0.3 mg/ml and diluted to 0.5 μg/ml in M9 buffer just before use. Mixtures of young adults and L4 larvae of tm234(ben-1);tm246(vps-45)-background Ex lines described above (cultured at 20°C) were collected from the NGM agar plates and incubated for 1 h at room temperature in the dark at 0.5 μg/ml of TMP. The animals were irradiated with 365 nm UV with a UV hand-monitor (UVP Inc.) at 200 J/cm2. The intensities of the UV light were calibrated with a UV luminometer (UVP Inc.) and controlled by the exposure time. UV/TMP-treated worms were plated on NGM agar dishes and allowed to lay eggs at 20°C. After 24 h, adults and larvae were washed off to remove all the animals fertilized before treatment, and incubated at 20°C for another 24 h, while F1 animals hatched. F1 animals were collected, washed three times with M9 buffer, and plated onto NGM agar plates containing 10 μg/ml of benzimidazole (Wako) (approximately 2000 animals/9-cm plate) and cultured at 20°C for 6 to 7 days. Because tm246(vps-45) mutants exhibit a temperature-sensitive phenotype, only tm246 mutants carrying vps-45 rescue transgenes can survive at 20°C (positive selection). In contrast, because ben-1(tm234) mutants are resistant to benzimidazole, tm234 mutants carrying ben-1 transgenes are sensitive and unable to survive on benzimidazole-containing plates (negative selection). Transformed animals were cloned and further cultured on benzimidazole-containing plates over several generations. To ensure that each strain was established independently, only one transformant was picked up from each selection plate. Transformants were selected by PCR#A and PCR#B (as shown in Figure 1), which amplify the 5' and 3' regions of HBG-LoxP-vps-45-LoxP, respectively. Primers used are listed in Additional file 4, Table S2.
Genomic-excision using Cre recombinase mRNA
Cre recombinase cDNA was amplified from AxCANCre (TaKaRa)  and cloned into the pGEMT vector. Cre recombinase RNA was synthesized in vitro using a mMESSAGE mMACHINE T7 kit (Ambion). Synthesized RNA was purified by phenol/chloroform extraction followed by isoamyl alcohol precipitation. Poly-A tailing was performed using Yeast Poly(A) Polymerase (74225Y, affymetrix). Poly-A tailed RNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Cre recombinase mRNA was injected at 1 μg/ml along with P myo-2 ::venus as an injection marker (67 ng/ml) into integrant animals carrying a floxed vps-45 mini gene. F1 animals were genotyped by PCR#1 to detect Cre-mediated excision. F2 self-progenies from heterozygotes for the Cre-excised allele were genotyped to obtain homozygotes for the Cre-excised allele. Homozygous lines were further tested by PCR#2 to detect non-excised allele (as shown in Figure 2). Primers used are listed in Additional file 4, Table S2.
Genome DNA was isolated from adult animals using DNeasy Tissue & Blood kit (QIAGEN). Quantitative PCR was performed in a 7500 Real-time Thermal cycler (Applied Biosystems) using the Power SYBR master mix (Applied Biosystems) with the following parameters: 95°C for 10 min and 40 cycles of 95°C for 5 s, 55°C for 10 s and 72°C for 34 s. All data were normalized to the act-2 gene. Primers were designed within an exon for each gene using the Primer3 software. The primers used are listed in Additional file 4, Table S2.
To map insertion sites, SNPs between the Hawaiian strain CB4856  and the parent strain Bristol N2, were used. Worm lysis and SNP mapping were based on the procedures described previously  with some modification. Briefly, N2-backround strains of interest were outcrossed 8 to 12 times with CB4856 and assayed for linkage between the HBG-loxP sequence (detected by PCR#1) and SNPs.
Differential interference contrast and fluorescence images were obtained using a BX51 microscope equipped with a DP30BW CCD camera (Olympus Optical Co., Ltd).
We thank Y Seyama for technical support; Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN, Wako, Saitama, Japan) for providing the Venus gene; and the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, USA; supported by the National Institutes of Health-National Center for Research Resources) for providing some C. elegans strains.
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