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Molecular genotyping of the mouse scid allele
Journal of Immunological Methods 260 (2002) 303 – 304 www.elsevier.com/locate/jim
Protocol
Molecular genotyping of the mouse scid allele Amy L. Sealey, Nicole K. Hobbs, Edward E. Schmidt * Veterinary Molecular Biology, Marsh Laboratories, Montana State University, Bozeman, MT 59717, USA Received 7 September 2001; accepted 11 September 2001
Abstract Severe combined immunodeficiency (SCID) mice, which lack mature B- and T-cells, provide an important system for studies on immunity and disease. However, since the scid mutation is a single T-to-A transversion, genotypic analysis can be problematic. We have developed a rapid and simple sequence-based analysis that provides identification of both the scid and the wild-type (+) allele. This allows unequivocal identification of scid/scid, scid/+ and +/+ individuals. The method is of greatest utility for discerning scid/+ from +/+ animals during genetic complementation studies, but may be of value for routine SCID colony control as well. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecular genotyping; Severe combined immunodeficiency mice; Mutation
Severe combined immunodeficiency (SCID) mice are homozygous for a mutation (scid) in the catalytic subunit of a DNA-dependent protein kinase, DNAPKcs (Blunt et al., 1996). As a result, they cannot complete V(D)J recombination and lack mature B-cells, T-cells, and antibody (Bosma and Carroll, 1991). Although the phenotypic consequence is severe, the mutation is subtle—a single T-to-A transversion (Blunt et al., 1996). Therefore, SCID mice are generally identified phenotypically, such as by their lack of serum IgG (Bosma and Carroll, 1991). Because the allele is recessive, scid/+ heterozygotes cannot be phenotypically discerned from +/+ animals (Fig. 1A). Many uses of SCID mice are performed on colonies of pure scid/ scid mice; however, genetic complementation studies yield F2 and later generations where littermates may be scid/scid, scid/+, or +/+. Although scid/+ and +/+ *
Corresponding author. Tel.: +1-406-994-6375; fax: +1-406994-4303. E-mail address: [email protected] (E.E. Schmidt).
animals can be retroactively inferred by breeding and assaying F3 phenotypes, direct molecular analysis is months faster and prevents unnecessary breeding. The scid mutation creates a cleavage site for the restriction enzyme Alu I in exon 85 of the DNAPKcs gene, which formed the basis of a previous molecular analysis (Blunt et al., 1996). Briefly, a region containing the scid mutation is amplified by PCR, digested with Alu I, and assayed by gel electrophoresis. Cleavage is indicative of a mutant allele, whereas wild-type alleles remain uncleaved. However, this assay can be problematic because failed or partial Alu I digestion could cause misidentification of mutant alleles as wild type. A sequence-based assay, we reasoned, would give a unique identifier for each allele, which could not be confounded. Genomic DNA was prepared by overnight digestion at 55 C in 390 ml of 50 mM Tris, pH. 7.5, 100 mM EDTA, 100 mM NaCl, 1% SDS, 100 mg/ml proteinase K of tail tips ( 0.5 cm long) harvested at weaning. Proteins and SDS were precipitated and removed by
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 1 ) 0 0 5 2 4 - 5
304
A.L. Sealey et al. / Journal of Immunological Methods 260 (2002) 303–304
Fig. 1. Phenotypic and genotypic analysis of SCID mice. (A) Serum IgG levels. Indicated dilutions of serum harvested from tail bleeds at weaning were spotted onto nitrocellulose filters. IgG was detected by chemiluminescence using the Pierce Supersignal kit and peroxidase-conjugated anti-mouse IgG by the manufacturer’s recommended protocols. Genotypes indicated at right were determined retroactively on tail DNA. (B) PCR of region surrounding the scid mutation. Crude tail DNA was amplified and analyzed by gel electrophoresis as described in the text. All 10 samples gave strong reliable sequence data (not shown). (C) Sequence analysis of scid mutation in mice. Representative scans of the region encompassing the scid mutation, as described in the text. The site of the scid mutation is indicated in bold font.
the addition of 260 ml 5 M NaCl, incubation on ice of 15 min, and centrifugation. Supernatant was transferred to tubes containing 900 ml of absolute ethanol to precipitate nucleic acids. Genomic DNA was spooled out on pipettor tips and transferred into tubes containing 200 ml TE. After DNA was dissolved, 0.2 ml of this was amplified directly in 25 ml PCR reactions using a 32-cycle PCR reaction with Taq polymerase, an annealing temperature of 54.8 C, and an elongation time of 1 min 20 s. PCR primers were designed to create a 370-bp product spanning from intron 84 (sense primer 5VGCTGTTCCAGTTATAGATCT-3V) to intron 85 (antisense primer 5V-AGCCGCCCTAAGAGTCACTTTC-3V). A portion (80%) of each PCR reaction was analyzed on agarose gels to ensure amplification (Fig. 1B), and a portion (10%) was used for direct sequencing of the PCR product. Briefly, to eliminate excess PCR primers, 2.5 ml of the PCR
reaction was added to tubes containing 11 ml of 0.4 M NaClO4, 44% isopropanol, samples were mixed, and PCR product was collected by centrifugation at room temperature. The supernatant was removed, the pellet was resuspended in 7 ml TE, and the product was subjected to automated sequencing by standard protocols on ABI 310 or ABI 377 sequencers using 3.2 pmol of the sense primer. Sequencing chromatograms were used to identify which alleles were present (Fig. 1C). Our approach of directly sequencing PCR products amplified from crude genomic tail DNA will likely have applications on other strains of mice. We have retroactively genotyped DNA samples stored for over a year at 4 C with undiminished reliability. Most importantly, the few reactions that have failed ( 2%) have provided no sequence rather than a mistaken genotype. It is nearly impossible for a scid allele to be misidentified as wild type, or vice versa, by this method (Fig. 1C). Although we developed this assay for ongoing genetic complementation studies, the ease and reliability of the procedure has led us to adopt it for routine genetic screening of our SCID colony as well.
Acknowledgements The authors thank Tammy Frerk for animal care and husbandry. Supported by a Basil O’Connor New Investigator Award from the March of Dimes Foundation, an appointment to the Montana Agricultural Experiment Station, UDSA Animal Health Formula Funds, and grants from NSF and NSF-MONTS to EES. ALS was supported by an NSF/IGERT award to the MSU Program in Complex Biological Systems. MAES/MSU-College of Agriculture manuscript #2001-52.
References Blunt, T., Gell, D., Fox, M., Taccioli, G.E., Lehmann, A.R., Jackson, S.P., Jeggo, P.A., 1996. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc. Natl. Acad. Sci. U. S. A. 93, 10285 – 10290. Bosma, M.J., Carroll, A.M., 1991. The SCID mouse mutant: definition, characterization, and potential uses. Annu. Rev. Immunol. 9, 323 – 350.
Protocol
Molecular genotyping of the mouse scid allele Amy L. Sealey, Nicole K. Hobbs, Edward E. Schmidt * Veterinary Molecular Biology, Marsh Laboratories, Montana State University, Bozeman, MT 59717, USA Received 7 September 2001; accepted 11 September 2001
Abstract Severe combined immunodeficiency (SCID) mice, which lack mature B- and T-cells, provide an important system for studies on immunity and disease. However, since the scid mutation is a single T-to-A transversion, genotypic analysis can be problematic. We have developed a rapid and simple sequence-based analysis that provides identification of both the scid and the wild-type (+) allele. This allows unequivocal identification of scid/scid, scid/+ and +/+ individuals. The method is of greatest utility for discerning scid/+ from +/+ animals during genetic complementation studies, but may be of value for routine SCID colony control as well. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecular genotyping; Severe combined immunodeficiency mice; Mutation
Severe combined immunodeficiency (SCID) mice are homozygous for a mutation (scid) in the catalytic subunit of a DNA-dependent protein kinase, DNAPKcs (Blunt et al., 1996). As a result, they cannot complete V(D)J recombination and lack mature B-cells, T-cells, and antibody (Bosma and Carroll, 1991). Although the phenotypic consequence is severe, the mutation is subtle—a single T-to-A transversion (Blunt et al., 1996). Therefore, SCID mice are generally identified phenotypically, such as by their lack of serum IgG (Bosma and Carroll, 1991). Because the allele is recessive, scid/+ heterozygotes cannot be phenotypically discerned from +/+ animals (Fig. 1A). Many uses of SCID mice are performed on colonies of pure scid/ scid mice; however, genetic complementation studies yield F2 and later generations where littermates may be scid/scid, scid/+, or +/+. Although scid/+ and +/+ *
Corresponding author. Tel.: +1-406-994-6375; fax: +1-406994-4303. E-mail address: [email protected] (E.E. Schmidt).
animals can be retroactively inferred by breeding and assaying F3 phenotypes, direct molecular analysis is months faster and prevents unnecessary breeding. The scid mutation creates a cleavage site for the restriction enzyme Alu I in exon 85 of the DNAPKcs gene, which formed the basis of a previous molecular analysis (Blunt et al., 1996). Briefly, a region containing the scid mutation is amplified by PCR, digested with Alu I, and assayed by gel electrophoresis. Cleavage is indicative of a mutant allele, whereas wild-type alleles remain uncleaved. However, this assay can be problematic because failed or partial Alu I digestion could cause misidentification of mutant alleles as wild type. A sequence-based assay, we reasoned, would give a unique identifier for each allele, which could not be confounded. Genomic DNA was prepared by overnight digestion at 55 C in 390 ml of 50 mM Tris, pH. 7.5, 100 mM EDTA, 100 mM NaCl, 1% SDS, 100 mg/ml proteinase K of tail tips ( 0.5 cm long) harvested at weaning. Proteins and SDS were precipitated and removed by
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 1 ) 0 0 5 2 4 - 5
304
A.L. Sealey et al. / Journal of Immunological Methods 260 (2002) 303–304
Fig. 1. Phenotypic and genotypic analysis of SCID mice. (A) Serum IgG levels. Indicated dilutions of serum harvested from tail bleeds at weaning were spotted onto nitrocellulose filters. IgG was detected by chemiluminescence using the Pierce Supersignal kit and peroxidase-conjugated anti-mouse IgG by the manufacturer’s recommended protocols. Genotypes indicated at right were determined retroactively on tail DNA. (B) PCR of region surrounding the scid mutation. Crude tail DNA was amplified and analyzed by gel electrophoresis as described in the text. All 10 samples gave strong reliable sequence data (not shown). (C) Sequence analysis of scid mutation in mice. Representative scans of the region encompassing the scid mutation, as described in the text. The site of the scid mutation is indicated in bold font.
the addition of 260 ml 5 M NaCl, incubation on ice of 15 min, and centrifugation. Supernatant was transferred to tubes containing 900 ml of absolute ethanol to precipitate nucleic acids. Genomic DNA was spooled out on pipettor tips and transferred into tubes containing 200 ml TE. After DNA was dissolved, 0.2 ml of this was amplified directly in 25 ml PCR reactions using a 32-cycle PCR reaction with Taq polymerase, an annealing temperature of 54.8 C, and an elongation time of 1 min 20 s. PCR primers were designed to create a 370-bp product spanning from intron 84 (sense primer 5VGCTGTTCCAGTTATAGATCT-3V) to intron 85 (antisense primer 5V-AGCCGCCCTAAGAGTCACTTTC-3V). A portion (80%) of each PCR reaction was analyzed on agarose gels to ensure amplification (Fig. 1B), and a portion (10%) was used for direct sequencing of the PCR product. Briefly, to eliminate excess PCR primers, 2.5 ml of the PCR
reaction was added to tubes containing 11 ml of 0.4 M NaClO4, 44% isopropanol, samples were mixed, and PCR product was collected by centrifugation at room temperature. The supernatant was removed, the pellet was resuspended in 7 ml TE, and the product was subjected to automated sequencing by standard protocols on ABI 310 or ABI 377 sequencers using 3.2 pmol of the sense primer. Sequencing chromatograms were used to identify which alleles were present (Fig. 1C). Our approach of directly sequencing PCR products amplified from crude genomic tail DNA will likely have applications on other strains of mice. We have retroactively genotyped DNA samples stored for over a year at 4 C with undiminished reliability. Most importantly, the few reactions that have failed ( 2%) have provided no sequence rather than a mistaken genotype. It is nearly impossible for a scid allele to be misidentified as wild type, or vice versa, by this method (Fig. 1C). Although we developed this assay for ongoing genetic complementation studies, the ease and reliability of the procedure has led us to adopt it for routine genetic screening of our SCID colony as well.
Acknowledgements The authors thank Tammy Frerk for animal care and husbandry. Supported by a Basil O’Connor New Investigator Award from the March of Dimes Foundation, an appointment to the Montana Agricultural Experiment Station, UDSA Animal Health Formula Funds, and grants from NSF and NSF-MONTS to EES. ALS was supported by an NSF/IGERT award to the MSU Program in Complex Biological Systems. MAES/MSU-College of Agriculture manuscript #2001-52.
References Blunt, T., Gell, D., Fox, M., Taccioli, G.E., Lehmann, A.R., Jackson, S.P., Jeggo, P.A., 1996. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc. Natl. Acad. Sci. U. S. A. 93, 10285 – 10290. Bosma, M.J., Carroll, A.M., 1991. The SCID mouse mutant: definition, characterization, and potential uses. Annu. Rev. Immunol. 9, 323 – 350.