An in vivo functional genetic screen for suppressors of the Rag1^−/− T-cell defect

Abstract

Functional genetic screens on mutant backgrounds have been successfully used in lower organisms to investigate biological processes. However, few identical screens have been performed in mice. Recombinase activating gene-1 deficient (Rag1^−/−) mice have a severe T-cell developmental block owing to lack of rearrangement of their T-cell receptor (TCR) genes. Using a retroviral cDNA library derived from wild-type embryonic thymocytes we performed a suppressor screen in Rag1^−/− hematopoietic cells and recovered TCRβ. This is the first demonstration that targeted genetic screens are feasible using transduced primary cells in vivo. Consequently, this technique can be used to interrogate multiple blood lineages using diverse hematopoietic mouse mutants.

Introduction

Genetic screens are powerful tools for dissecting important signaling pathways as demonstrated by functional screens performed in established model organisms such as yeast and Drosophila. With a high degree of genetic conservation between mouse and human, the mouse is an ideal model for genetic screening. The sequencing of the mouse genome and the burgeoning number of mutant mouse models has made genetic screens in the mouse more accessible. Since the discovery of ethylnitrosurea as an efficient mutagen in spermatogonial stem cells, mutagenesis screens in mice have been utilized as a means to characterize biological processes (Kile and Hilton, 2005, Russell and Kelly, 1982).

Recently, this mutagenesis approach identified c-Myb as a dominant suppressor of the thrombocytopenic Mpl^−/− phenotype, establishing the first modifier screen in the mouse (Carpinelli et al., 2004). However, few laboratories possess the resources to carry out such time consuming and financially expensive screens. Here, we propose an alternative approach by using a retroviral cDNA library to functionally screen for suppression of a mouse mutant phenotype. Retroviral cDNA libraries have been successfully used in cell lines to identify oncogenes and components of signal transduction pathways (Choi et al., 2005, Passioura et al., 2005, Zhong et al., 1994). Using retroviral technology and flow cytometry, we have designed a suppressor screen for genes that can overcome defective T-cell development in the Rag1^−/− mouse.

The stages of αβ T-cell development in the mouse thymus are characterized by expression of cell surface markers CD4 and CD8. T-cell precursors are initially CD4⁻CD8⁻ (double negative; DN), and can be further developmentally categorized by expression of CD25 and CD44 (Godfrey et al., 1993). The CD25⁻CD44⁺ (DN1), CD25⁺CD44⁺ (DN2), CD25⁺CD44⁻ (DN3) and CD25⁻CD44⁻ (DN4) developmental stages correspond to increasing commitment to the T-cell lineage. CD4⁺CD8⁺ (double positive; DP) and functionally mature single positive (SP) CD4⁻CD8⁺ and CD4⁺CD8⁻ cells arise from DN cells (Fig. 1) (Michie and Zuniga-Pflucker, 2002). This DN to DP transition is accompanied by an exponential increase in cell number and as such is a prime target for deregulation (Fehling and von Boehmer, 1997). Uncontrolled proliferation of DP cells in T-cell acute lymphoblastic leukemias caused by the translocation of wild-type genes such as SCL/TAL1, NOTCH1 and HOX11 into T-cell receptor (TCR) loci demonstrates the importance of regulating the DN–DP transition (Ellisen et al., 1991, Hatano et al., 1991, Hershfield et al., 1984).

In the first of a series of checkpoints regulating T-cell development, DN3 cells must express a functionally rearranged β chain of the T-cell antigen receptor (TCRβ) as a component of the pre-TCR complex with pre-Tα and CD3 to mature to the DP stage (Michie and Zuniga-Pflucker, 2002). This process is known as β-selection and is dependent upon TCRβ rearrangement mediated by the recombinase activating genes (Rag) Rag1 and Rag2 (Mombaerts et al., 1992b, Shinkai et al., 1992). Thus, T-cell precursors in Rag1^−/− and Rag2^−/− mice fail pre-TCR-dependent β-selection and are developmentally arrested at the CD25⁺CD44⁻ DN3 stage of T-cell ontogeny (Mombaerts et al., 1992b). DN thymocytes in Rag1^−/− or Rag2^−/− mice can only bypass this developmental arrest to become DP cells when wild-type transgenes such as Tcrβ, Pim1, Cd28, Nfκb or oncogenes such as Ha-ras^V12 (Jacobs et al., 1999, Shinkai et al., 1993, Shinkai et al., 1992, Swat et al., 1996, Voll et al., 2000, Williams et al., 2005) and intracellular Notch1 (ICN1) are introduced (Campese et al., 2006 and this report).

We have designed a suppressor screen for genes that overcome the T-cell developmental block in Rag1^−/− hematopoietic cells. cDNAs identified by this screen are likely to have a significant role in T-cell development. Lethally irradiated C57BL/6J recipients were reconstituted with library-transduced Rag1^−/− bone marrow cells and DP cells were observed in the thymus of some recipients. One library cDNA that successfully overcame the DN block in Rag1^−/− precursors was identified as TCRβ. The DP phenotype was reproduced in fetal thymic organ culture (FTOC) when TCRβ-transduced Rag1^−/− fetal liver cells were used to reconstitute irradiated Rag1^−/− fetal thymi. Additionally, when re-introduced into Rag1^−/− recipients, TCRβ-transduced Rag1^−/− bone marrow was able to rescue the Rag1^−/− DN phenotype. Recovery of TCRβ from the wild-type thymocyte library validates the use of the screen to isolate genes crucial for T-cell development. This is the first in vivo application of a functional genetic screen that uses transduced primary cells, with our findings supporting the ease and effectiveness of this experimental approach.