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.
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References
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
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
Orphanides G, Reinberg D (2002) A unified theory of gene expression. Cell 108(4):439–451
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
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
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
Crotty S (2011) Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29:621–663. doi:10.1146/annurev-immunol-031210-101400
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
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
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
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
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
Crotty S (2012) The 1-1-1 fallacy. Immunol Rev 247(1):133–142. doi:10.1111/j.1600-065X.2012.01117.x
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
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
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
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
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
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
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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
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DOI: https://doi.org/10.1007/978-1-4939-2498-1_5
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