Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Genetic knockouts and knockins in human somatic cells

Nature Protocols volume 2, pages 2734–2746 (2007)Cite this article

Abstract

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the generation of knockout and knockin alleles by homologous recombination.
Figure 2: Assembly of an rAAV targeting construct.
Figure 3: Synthesis of HAs.
Figure 4: Screen for homologous integration.

Similar content being viewed by others

References

  1. Bunz, F. Human cell knockouts. Curr. Opin. Oncol. 14, 73–78 (2002).

    Article  PubMed  Google Scholar 

  2. Cassman, M. Computational biology. Counting on the neuron. Science 300, 756–757 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H. & Williams, B.R. Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Bridge, A.J., Pebernard, S., Ducraux, A., Nicoulaz, A.L. & Iggo, R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34, 263–264 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Vogelstein, B. et al. Genetic alterations during colorectal-tumor development. N. Engl. J. Med. 319, 525–532 (1988).

    Article  CAS  PubMed  Google Scholar 

  8. Vogelstein, B. & Kinzler, K.W. The multistep nature of cancer. Trends Genet. 9, 138–141 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Wang, T.L. et al. Prevalence of somatic alterations in the colorectal cancer cell genome. Proc. Natl. Acad. Sci. USA 99, 3076–3080 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sjoblom, T. et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006).

    PubMed  Google Scholar 

  11. Lengauer, C., Kinzler, K.W. & Vogelstein, B. Genetic instability in colorectal cancers. Nature 386, 623–627 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Lengauer, C., Kinzler, K.W. & Vogelstein, B. Genetic instabilities in human cancers. Nature 396, 643–649 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Parsons, R. et al. Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell 75, 1227–1236 (1993).

    Article  CAS  PubMed  Google Scholar 

  14. Montrose-Rafizadeh, C. et al. Gene targeting of a CFTR allele in HT29 human epithelial cells. J. Cell Physiol. 170, 299–308 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Itzhaki, J.E., Gilbert, C.S. & Porter, A.C. Construction by gene targeting in human cells of a 'conditional' CDC2 mutant that rereplicates its DNA. Nat. Genet. 15, 258–265 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Hirata, R., Chamberlain, J., Dong, R. & Russell, D.W. Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat. Biotechnol. 20, 735–738 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Karakas, B. et al. Interleukin-1 alpha mediates the growth proliferative effects of transforming growth factor-beta in p21 null MCF-10A human mammary epithelial cells. Oncogene 25, 5561–5569 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Chamberlain, J.R. et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303, 1198–1201 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Kohli, M., Rago, C., Lengauer, C., Kinzler, K.W. & Vogelstein, B. Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res. 32, e3 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Cummins, J.M. et al. Tumour suppression: disruption of HAUSP gene stabilizes p53. Nature 428 1 p following 486 (2004).

    Article  PubMed  Google Scholar 

  21. Sedivy, J.M. & Dutriaux, A. Gene targeting and somatic cell genetics—a rebirth or a coming of age? Trends Genet 15, 88–90 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  PubMed  Google Scholar 

  23. Yanez, R.J. & Porter, A.C. Therapeutic gene targeting. Gene Ther. 5, 149–159 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Topaloglu, O., Hurley, P.J., Yildirim, O., Civin, C.I. & Bunz, F. Improved methods for the generation of human gene knockout and knockin cell lines. Nucleic Acids Res. 33, e158 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Russell, D.W. & Hirata, R.K. Human gene targeting by viral vectors. Nat. Genet. 18, 325–330 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Choi, T., Huang, M., Gorman, C. & Jaenisch, R. A generic intron increases gene expression in transgenic mice. Mol. Cell. Biol. 11, 3070–3074 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huang, M.T. & Gorman, C.M. Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 18, 937–947 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hellen, C.U. & Sarnow, P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Park, B.H., Vogelstein, B. & Kinzler, K.W. Genetic disruption of PPARdelta decreases the tumorigenicity of human colon cancer cells. Proc. Natl. Acad. Sci. USA 98, 2598–2603 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jallepalli, P.V. et al. Securin is required for chromosomal stability in human cells. Cell 105, 445–457 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Hanson, K.D. & Sedivy, J.M. Analysis of biological selections for high-efficiency gene targeting. Mol. Cell. Biol. 15, 45–51 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Friedel, R.H. et al. Gene targeting using a promoterless gene trap vector ('targeted trapping') is an efficient method to mutate a large fraction of genes. Proc. Natl. Acad. Sci. USA 102, 13188–13193 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Porteus, M.H., Cathomen, T., Weitzman, M.D. & Baltimore, D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol. Cell. Biol. 23, 3558–3565 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wilsker, D. & Bunz, F. Loss of ataxia telangiectasia mutated- and Rad3-related function potentiates the effects of chemotherapeutic drugs on cancer cell survival. Mol. Cancer Ther. 6, 1406–1413 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, P., Yu, J. & Zhang, L. The nuclear function of p53 is required for PUMA-mediated apoptosis induced by DNA damage. Proc. Natl. Acad. Sci. USA 104, 4054–4059 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Larochelle, S. et al. Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol. Cell 25, 839–850 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim, J.S., Lee, C., Bonifant, C.L., Ressom, H. & Waldman, T. Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA. Mol. Cell. Biol. 27, 662–677 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Hurley, P.J., Wilsker, D. & Bunz, F. Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation. Oncogene 26, 2535–2542 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Burkard, M.E. et al. Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells. Proc. Natl. Acad. Sci. USA 104, 4383–4388 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gallmeier, E. et al. Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options. Gastroenterology 130, 2145–2154 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Dang, L.H. et al. CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48. Oncogene 25, 2264–2272 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Dang, D.T. et al. Hypoxia-inducible factor-1alpha promotes nonhypoxia-mediated proliferation in colon cancer cells and xenografts. Cancer Res. 66, 1684–1936 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Cunningham, S.C. et al. Targeted deletion of MKK4 in cancer cells: a detrimental phenotype manifests as decreased experimental metastasis and suggests a counterweight to the evolution of tumor-suppressor loss. Cancer Res. 66, 5560–5564 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Samuels, Y. et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7, 561–573 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Papi, M., Berdougo, E., Randall, C.L., Ganguly, S. & Jallepalli, P.V. Multiple roles for separase auto-cleavage during the G2/M transition. Nat. Cell Biol. 7, 1029–1035 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Dang, D.T. et al. Glutathione I-transferase pi1 promotes tumorigenicity in HCT116 human colon cancer cells. Cancer Res. 65, 9485–9494 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Cummins, J.M. et al. X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells. Cancer Res. 64, 3006–3008 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Torrance, C.J., Agrawal, V., Vogelstein, B. & Kinzler, K.W. Use of isogenic human cancer cells for high-throughput screening and drug discovery. Nat. Biotechnol. 19, 940–945 (2001).

    Article  CAS  PubMed  Google Scholar 

  49. Arena, S., Pisacane, A., Mazzone, M., Comoglio, P.M. & Bardelli, A. Genetic targeting of the kinase activity of the Met receptor in cancer cells. Proc. Natl. Acad. Sci. USA 104, 11412–11417 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  PubMed  Google Scholar 

  51. Dong, J.Y., Fan, P.D. & Frizzell, R.A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther. 7, 2101–2112 (1996).

    Article  CAS  PubMed  Google Scholar 

  52. Hirata, R.K. & Russell, D.W. Design and packaging of adeno-associated virus gene targeting vectors. J. Virol. 74, 4612–4620 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Vogelstein, B. & Gillespie, D. Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615–619 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Vogelstein, B. Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column. Anal. Biochem. 160, 115–118 (1987).

    Article  CAS  PubMed  Google Scholar 

  55. Chan, T.A., Wang, Z., Dang, L.H., Vogelstein, B. & Kinzler, K.W. Targeted inactivation of CTNNB1 reveals unexpected effects of beta-catenin mutation. Proc. Natl. Acad. Sci. USA 99, 8265–8270 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chan, T.A., Hwang, P.M., Hermeking, H., Kinzler, K.W. & Vogelstein, B. Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev. 14, 1584–1588 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Chan, T.A., Hermeking, H., Lengauer, C., Kinzler, K.W. & Vogelstein, B. 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature 401, 616–620 (1999).

    Article  CAS  PubMed  Google Scholar 

  58. Hwang, P.M. et al. Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells. Nat. Med. 7, 1111–1117 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Ramirez-Solis, R., Davis, A.C. & Bradley, A. Gene targeting in embryonic stem cells. Methods Enzymol. 225, 855–878 (1993).

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Sidney Kimmel Comprehensive Cancer Center, Cellular and Molecular Medicine Program, Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins University School of Medicine, 1550 Orleans Street CRB2-453, Baltimore, 21231, Maryland, USA

    Carlo Rago & Bert Vogelstein

  2. Sidney Kimmel Comprehensive Cancer Center, Cellular and Molecular Medicine Program, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 1550 Orleans Street CRB2-453, Baltimore, 21231, Maryland, USA

    Carlo Rago & Bert Vogelstein

  3. Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, Cellular and Molecular Medicine Program, Johns Hopkins University School of Medicine, 1550 Orleans Street CRB2-453, Baltimore, 21231, Maryland, USA

    Fred Bunz

Authors
  1. Carlo Rago
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bert Vogelstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fred Bunz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fred Bunz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rago, C., Vogelstein, B. & Bunz, F. Genetic knockouts and knockins in human somatic cells. Nat Protoc 2, 2734–2746 (2007). https://doi.org/10.1038/nprot.2007.408

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.408

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing