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In Vivo Rescue Assay of RhoA-Deficient Hematopoietic Stem and Progenitor Cells

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Book cover Rho GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1821))

Abstract

RhoA is a founding member of Rho GTPase family and is a well-recognized regulator of actin cytoskeleton dynamics. Signal pathways transduced by RhoA are involved in cell migration, polarity, morphogenesis, proliferation, survival, and cell fate decision. Conditional gene targeting of Rhoa in murine blood system induces acute hematopoietic failure due to the loss of multilineage hematopoietic progenitor cells (HPC) caused by a cytokinesis defect and necrosis. Here we describe a method to conditionally induce Rhoa gene knockout in murine blood cells and a rescue by exogenous RhoA expression with lentivirus in HPCs, an approach that has general applicability in studying in vivo function of Rho GTPases and their regulators/effectors by gene targeting.

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References

  1. Hu S, Qian M, Zhang H, Guo Y, Yang J, Zhao X, He H, Lu J, Pan J, Chang M, Du G, Lin TN, Kham SK, Quah TC, Ariffin H, Tan AM, Cheng Y, Li C, Yeoh AE, Pui CH, Skanderup AJ, Yang JJ (2017) Whole-genome noncoding sequence analysis in T-cell acute lymphoblastic leukemia identifies oncogene enhancer mutations. Blood 129:3264–3268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Makishima H, Yoshizato T, Yoshida K, Sekeres MA, Radivoyevitch T, Suzuki H, Przychodzen B, Nagata Y, Meggendorfer M, Sanada M, Okuno Y, Hirsch C, Kuzmanovic T, Sato Y, Sato-Otsubo A, LaFramboise T, Hosono N, Shiraishi Y, Chiba K, Haferlach C, Kern W, Tanaka H, Shiozawa Y, Gómez-Seguí I, Husseinzadeh HD, Thota S, Guinta KM, Dienes B, Nakamaki T, Miyawaki S, Saunthararajah Y, Chiba S, Miyano S, Shih LY, Haferlach T, Ogawa S, Maciejewski JP (2017) Dynamics of clonal evolution in myelodysplastic syndromes. Nat Genet 49:204–212

    Article  CAS  PubMed  Google Scholar 

  3. Ding LW, Sun QY, Tan KT, Chien W, Mayakonda A, Yeoh AEJ, Kawamata N, Nagata Y, Xiao JF, Loh XY, Lin DC, Garg M, Jiang YY, Xu L, Lim SL, Liu LZ, Madan V, Sanada M, Fernández LT, Hema Preethi SS, Lill M, Kantarjian HM, Kornblau SM, Miyano S, Liang DC, Ogawa S, Shih LY, Yang H, Koeffler HP (2017) Mutational landscape of pediatric acute lymphoblastic leukemia. Cancer Res 77:390–400

    Article  CAS  PubMed  Google Scholar 

  4. Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, Potter NE, Heuser M, Thol F, Bolli N, Gundem G, Van Loo P, Martincorena I, Ganly P, Mudie L, McLaren S, O'Meara S, Raine K, Jones DR, Teague JW, Butler AP, Greaves MF, Ganser A, Döhner K, Schlenk RF, Döhner H, Campbell PJ (2016) Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med 374:2209–2221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Taylor J, Xiao W, Abdel-Wahab O (2017) Diagnosis and classification of hematologic malignancies on the basis of genetics. Blood 130:410–423

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Patel BJ, Przychodzen B, Thota S, Radivoyevitch T, Visconte V, Kuzmanovic T, Clemente M, Hirsch C, Morawski A, Souaid R, Saygin C, Nazha A, Demarest B, LaFramboise T, Sakaguchi H, Kojima S, Carraway HE, Ogawa S, Makishima H, Sekeres MA, Maciejewski JP (2017) Genomic determinants of chronic myelomonocytic leukemia. Leukemia 31:2815–2833

    Article  CAS  PubMed  Google Scholar 

  7. Van Vlierberghe P, Ferrando A (2012) The molecular basis of T cell acute lymphoblastic leukemia. J Clin Invest 122:3398–3406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Geh E, Meng Q, Mongan M, Wang J, Takatori A, Zheng Y, Puga A, Lang RA, Xia Y (2011) Mitogen-activated protein kinase kinase kinase 1 (MAP 3K1) integrates developmental signals for eyelid closure. Proc Natl Acad Sci U S A 108:17349–17354

    Article  PubMed  PubMed Central  Google Scholar 

  9. Jackson B, Peyrollier K, Pedersen E, Basse A, Karlsson R, Wang Z, Lefever T, Ochsenbein AM, Schmidt G, Aktories K, Stanley A, Quondamatteo F, Ladwein M, Rottner K, van Hengel J, Brakebusch C (2011) RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes. Mol Biol Cell 22:593–605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Melendez J, Stengel K, Zhou X, Chauhan BK, Debidda M, Andreassen P, Lang RA, Zheng Y (2011) RhoA GTPase is dispensable for actomyosin regulation but is essential for mitosis in primary mouse embryonic fibroblasts. J Biol Chem 286:15132–15137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Pleines I, Hagedorn I, Gupta S, May F, Chakarova L, van Hengel J, Offermanns S, Krohne G, Kleinschnitz C, Brakebusch C, Nieswandt B (2012) Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood 119:1054–1063

    Article  CAS  PubMed  Google Scholar 

  12. Xiang SY, Vanhoutte D, Del Re DP, Purcell NH, Ling H, Banerjee I, Bossuyt J, Lang RA, Zheng Y, Matkovich SJ, Miyamoto S, Molkentin JD, Dorn GW, Brown JH (2011) RhoA protects the mouse heart against ischemia/reperfusion injury. J Clin Invest 121:3269–3276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang S, Zhou X, Lang RA, Guo F (2012) RhoA of the rho family small GTPases is essential for B lymphocyte development. PLoS One 7:e33773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zandvakili I, Lin Y, Morris JC, Zheng Y (2017) Rho GTPases: anti- or pro-neoplastic targets? Oncogene 36:3213–3222

    Article  CAS  PubMed  Google Scholar 

  15. Paul CD, Mistriotis P, Konstantopoulos K (2017) Cancer cell motility: lessons from migration in confined spaces. Nat Rev Cancer 17:131–140

    Article  CAS  PubMed  Google Scholar 

  16. Ghiaur G, Lee A, Bailey J, Cancelas JA, Zheng Y, Williams DA (2006) Inhibition of RhoA GTPase activity enhances hematopoietic stem and progenitor cell proliferation and engraftment. Blood 108:2087–2094

    Article  CAS  PubMed  Google Scholar 

  17. Zhou X, Florian MC, Arumugam P, Chen X, Cancelas JA, Lang R, Malik P, Geiger H, Zheng Y (2013) RhoA GTPase controls cytokinesis and programmed necrosis of hematopoietic progenitors. J Exp Med 210:2371–2385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. https://www.addgene.org/viral-vectors/retrovirus/retro-guide/

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Acknowledgments

This work was supported in part by US NIH grants R01CA193350 and R01DK104814 to Y.Z.

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Authors and Affiliations

  1. Cancer and Blood Diseases Institute, Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH, USA

    Xiongwei Cai, Xuan Zhou & Yi Zheng

  2. Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University, Chongqing, China

    Xiongwei Cai

Authors
  1. Xiongwei Cai
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  2. Xuan Zhou
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  3. Yi Zheng
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Corresponding author

Correspondence to Yi Zheng .

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Editors and Affiliations

  1. The Hull York Medical School, University of Hull, Hull, United Kingdom

    Francisco Rivero

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Cai, X., Zhou, X., Zheng, Y. (2018). In Vivo Rescue Assay of RhoA-Deficient Hematopoietic Stem and Progenitor Cells. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 1821. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8612-5_17

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  • DOI: https://doi.org/10.1007/978-1-4939-8612-5_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8611-8

  • Online ISBN: 978-1-4939-8612-5

  • eBook Packages: Springer Protocols

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