Published: Vol 7, Iss 23, Dec 5, 2017 DOI: 10.21769/BioProtoc.2627 Views: 8893
Reviewed by: Jia LiXi FengXiaoyi Zheng
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Abstract
Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models using retroviral gene transduction, xenograft, or Cre-mediated chromosomal translocation have been developed over the past twenty years. Particularly, a retroviral gene transduction-mediated murine leukemia model has been frequently used in the leukemia research field. Here, we describe the detailed protocol for this model.
Keywords: Mixed lineage leukemiaBackground
Gene rearrangements generate mixed-lineage leukemia (MLL) fusion genes, which cause highly aggressive acute leukemia. MLL-rearrangements are often associated with few additional genetic alterations and poor clinical outcomes (Andersson et al., 2015). Wild-type MLL enhances and maintains the expression of a subset of genes, including homeobox (Hox) genes, to stimulate the expansion of immature progenitors (Jude et al., 2007). The expression of Hoxa9 and Meis1 is highest in the immature progenitor/stem cell fraction, but gradually declines as cells differentiate, and eventually diminishes in terminally-differentiated cell fractions (Somervaille and Cleary, 2006; Yokoyama et al., 2013). The MLL fusion protein constitutively up-regulates the expression of target genes, including Hoxa9 and Meis1, to immortalize immature progenitor cells and cause leukemia in vivo (Ayton and Cleary, 2003; Lavau et al., 1997). To date, more than 130 different MLL-rearrangements have been identified (Meyer et al., 2017). Two-thirds of MLL-rearranged leukemia cases are caused by fusion with a gene that is part of the AF4 family-ENL family-P-TEFb (AEP) complex (Yokoyama et al., 2010). The MLL fusion proteins constitutively form an MLL/AEP hybrid complex on the target chromatin (Okuda et al., 2014; Yokoyama et al., 2010), which further associates with the SL1 complex to activate RNA polymerase II-dependent transcription (Okuda et al., 2015 and 2016). AEP-mediated transactivation of MLL target genes transformed myeloid progenitors ex vivo, but did not cause leukemia in vivo, which suggested that other function is additionally required for in vivo leukemogenesis (Okuda et al., 2017). Recently, we showed that the ability to recruit the DOT1L complex is necessary to cause leukemia in vivo in addition to the ability to recruit AEP using in vivo leukemogenesis model. Thus, the combinatorial use of the in vivo leukemogenesis model and myeloid progenitor transformation assay is necessary to dissect the functional properties of oncogenes. In this protocol, we describe the in vivo leukemogenesis model using retroviral transduction in detail.
Materials and Reagents
Equipment
Software
Procedure
Schedule:
Day 1. Start culturing PLAT-E cells from freeze stock
Day 4. Replate PLAT-E cells for transfection
Day 5. Transfect PLAT-E cells
Day 6. Preparation of c-kit positive cells and irradiation of mice
Day 7. Transduction of retrovirus to c-kit positive cells and injection
Notes:
Data analysis
Recipes
Acknowledgments
This study was supported by JSPS KAKENHI grants to H.O. (number 17H07379) and A.Y. (number 16H05337). This protocol is based on a previous report by Lavau et al. (1997). The authors declare no conflict of interest.
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Okuda, H. and Yokoyama, A. (2017). In vivo Leukemogenesis Model Using Retrovirus Transduction. Bio-protocol 7(23): e2627. DOI: 10.21769/BioProtoc.2627.
Category
Cancer Biology > General technique > Animal models
Cell Biology > Cell isolation and culture > Transformation
Cancer Biology > Oncogenesis > Leukemogenesis
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