Skip to main content

Retroviral Vector Expression in TCR Transgenic CD4+ T Cells

  • Protocol
T follicular Helper Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1291))

Abstract

The regulation of gene expression is key to understand the function of genes of interest. To explore the biological functions of genes, transgenic knock-in or knockout technologies have served as invaluable tools. While recent advances in molecular biology have introduced new techniques (i.e., CRISPR mediated gene editing) (Cong et al., Science 339(6121):819–823, 2013; Wang et al., Cell 153(4):910–918, 2013) for the generation of transgenic mice in a relatively short period of time, it can still take a long time to test biological hypotheses from scratch to design how to generate knock-in or knockout mice. Here, we describe methods to manipulate gene expression in T cell receptor (TCR) transgenic CD4 T cells, which allow us to investigate gene functions in the study of differentiation pathways of follicular helper T (Tfh) cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. doi:10.1126/science.1231143

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. doi:10.1016/j.cell.2013.04.025

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Orphanides G, Reinberg D (2002) A unified theory of gene expression. Cell 108(4):439–451

    Article  CAS  PubMed  Google Scholar 

  4. Jojic V, Shay T, Sylvia K, Zuk O, Sun X, Kang J, Regev A, Koller D, Consortium IGP, Best AJ, Knell J, Goldrath A, Jojic V, Koller D, Shay T, Regev A, Cohen N, Brennan P, Brenner M, Kim F, Rao TN, Wagers A, Heng T, Ericson J, Rothamel K, Ortiz-Lopez A, Mathis D, Benoist C, Bezman NA, Sun JC, Min-Oo G, Kim CC, Lanier LL, Miller J, Brown B, Merad M, Gautier EL, Jakubzick C, Randolph GJ, Monach P, Blair DA, Dustin ML, Shinton SA, Hardy RR, Laidlaw D, Collins J, Gazit R, Rossi DJ, Malhotra N, Sylvia K, Kang J, Kreslavsky T, Fletcher A, Elpek K, Bellemare-Pelletier A, Malhotra D, Turley S (2013) Identification of transcriptional regulators in the mouse immune system. Nat Immunol 14(6):633–643. doi: 10.1038/ni.2587

  5. Pfeffer K, Mak TW (1994) Lymphocyte ontogeny and activation in gene targeted mutant mice. Annu Rev Immunol 12:367–411. doi:10.1146/annurev.iy.12.040194.002055

    Article  CAS  PubMed  Google Scholar 

  6. von Boehmer H (1990) Developmental biology of T cells in T cell-receptor transgenic mice. Annu Rev Immunol 8:531–556. doi:10.1146/annurev.iy.08.040190.002531

    Article  Google Scholar 

  7. Crotty S (2011) Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29:621–663. doi:10.1146/annurev-immunol-031210-101400

    Article  CAS  PubMed  Google Scholar 

  8. Johnston RJ, Poholek AC, Ditoro D, Yusuf I, Eto D, Barnett B, Dent AL, Craft J, Crotty S (2009) Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science 325(5943):1006–1010. doi:10.1126/science.1175870

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, Wang Y-H, Dong C (2009) Bcl6 mediates the development of T follicular helper cells. Science 325(5943):1001–1005. doi:10.1126/science.1176676

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Yu D, Rao S, Tsai LM, Lee SK, He Y, Sutcliffe EL, Srivastava M, Linterman M, Zheng L, Simpson N, Ellyard JI, Parish IA, Ma CS, Li Q-J, Parish CR, Mackay CR, Vinuesa CG (2009) The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment. Immunity 31(3):457–468. doi:10.1016/j.immuni.2009.07.002

    Article  CAS  PubMed  Google Scholar 

  11. Kroenke MA, Eto D, Locci M, Cho M, Davidson T, Haddad EK, Crotty S (2012) Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell differentiation. J Immunol 188(8):3734–3744. doi:10.4049/jimmunol.1103246

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Choi YS, Yang JA, Crotty S (2013) Dynamic regulation of Bcl6 in follicular helper CD4 T (Tfh) cells. Curr Opin Immunol 1–7. doi:10.1016/j.coi.2013.04.003

  13. Crotty S (2012) The 1-1-1 fallacy. Immunol Rev 247(1):133–142. doi:10.1111/j.1600-065X.2012.01117.x

    Article  PubMed  Google Scholar 

  14. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7(12):1063–1066. doi:10.1038/sj.gt.3301206

    Article  CAS  PubMed  Google Scholar 

  15. Ai H-W, Hazelwood KL, Davidson MW, Campbell RE (2008) Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat Methods 5(5):401–403. doi:10.1038/nmeth.1207

    Article  CAS  PubMed  Google Scholar 

  16. Johnston RJ, Choi YS, Diamond JA, Yang JA, Crotty S (2012) STAT5 is a potent negative regulator of TFH cell differentiation. J Exp Med 209(2):243–250. doi:10.1084/jem.20111174

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Chen R, Bélanger S, Frederick MA, Li B, Johnston RJ, Xiao N, Liu Y-C, Sharma S, Peters B, Rao A, Crotty S, Pipkin ME (2014) In vivo RNA interference screens identify regulators of antiviral CD4+ and CD8+ T cell differentiation. Immunity 41(2):325–338

    Article  CAS  PubMed  Google Scholar 

  18. Choi YS, Eto D, Yang JA, Lao C, Crotty S (2013) Cutting edge: STAT1 is required for IL-6-mediated Bcl6 induction for early follicular helper cell differentiation. J Immunol. doi:10.4049/jimmunol.1203032

    Google Scholar 

  19. Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol 70(8):5701–5705

    PubMed Central  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Youn Soo Choi .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Choi, Y.S., Crotty, S. (2015). Retroviral Vector Expression in TCR Transgenic CD4+ T Cells. In: Espéli, M., Linterman, M. (eds) T follicular Helper Cells. Methods in Molecular Biology, vol 1291. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2498-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2498-1_5

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2497-4

  • Online ISBN: 978-1-4939-2498-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics