Retroviral Transduction of Quiescent Murine Hematopoietic Stem Cells

Abstract

Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation, cell and gene therapy applications, and the development of leukemia models for research. Because the hematopoietic progeny carries the genetic information of HSCs and replenishes the blood and immune system, corrective gene transfer into HSCs provides an ideal therapeutic approach for many monogenic hematological diseases and a useful tool for studies of HSC function and blood formation in normal and malignant hematopoiesis. However, the efficiency of gene transfer into HSCs has been limited by several features of viral vectors, viral titer, methods of viral transduction, and the property of stem cell quiescence. In this chapter, we describe the production of retrovirus using murine stem cell virus (MSCV)-based retroviral vectors and purification and transduction of murine HSCs.

A KLF4-DYRK2–mediated pathway regulating self-renewal in CML stem cells

“…LSK CD150 1 cells were then transformed by retroviral transduction using the MSCV-IRES-GFP retrovirus carrying BCR-ABL1(p210) and GFP, sorted for GFP, and transplanted in small numbers (to mimic chronic disease development) into cytoablated wild-type mice with radioprotective bone marrow (Figure 1B). 37 In contrast to mice in the wild-type group, a significantly lower number of mice in the group transplanted with BCR-ABL1 1 Klf4 D/D cells displayed expansion of myeloid leukemia cells (CD11b 1 Gr1 1 ) (Figure 1C; supplemental Figure 1). Interestingly, the Klf4 D/D CML group showed slower disease progression and lower penetrance caused by myeloid leukemia (1/10) or B-cell leukemia (2/10), whereas the control group developed leukemia (7/10) more rapidly (Figure 1D-E).…”

“…LSK CD150 1 cells were plated on RetroNectin-coated plates and transduced with BCR-ABL1 retrovirus in the presence of Polybrene (8 mg/mL) by spinoculation (60 minutes at 456 relative centrifugal force). 37 Retroviruses were generated by cotransfection of the MSCV-IRES-GFP construct carrying the BCR-ABL1 (p210) complementary DNA and pEco packaging plasmid in 293T cells. After transduction (15%-22% GFP 1 ), the cells were cultured for 2 to 3 days in the presence of stem cell factor (100 ng/mL), interleukin-3 (6 ng/mL), and interleukin-6 (10 ng/ mL) in X-VIVO 15 medium, and GFP 1 cells were purified by cell sorting.…”