Optimized Retroviral Transduction Protocol Which Preserves the Primitive Subpopulation of Human Hematopoietic Cells
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- 作者：Alex Tonks ; Amanda J. Tonks ; Lorna Pearn ; Zulkhairi Mohamad ; Alan K. Burnett ; and Richard L. Darley
- 刊名：Biotechnology Progress
- 出版年：2005
- 出版时间：June 2005
- 年：2005
- 卷：21
- 期：3
- 页码：953 - 958
- 全文大小：258K
- 年卷期：v.21,no.3(June 2005)
- ISSN：1520-6033
文摘
Though both low-speed centrifugation and the use of fibronectin (Retronectin)fragments increase gene transduction efficiency, they still do not overcome the adverseeffects of the presence of virus-containing medium (VCM). In this study, we improvedtransduction efficiency of primitive human hematopoietic cells by optimizing theconditions for preadsorbing culture dishes with retrovirus using a centrifugationprotocol allowing subsequent infection to be carried out in the absence of VCM. Wealso demonstrate that preadsorbing tissue culture plates with retrovirus is dependenton the volume of VCM used for preadsorption and the length of centrifugation andthe type of plasticware used but not on the temperature of centrifugation (4-33 
C).Direct exposure of CD34+ target cells to VCM depletes the primitive CD34+CD38negsubpopulation by more than 30%, whereas the optimized VCM-free infection protocoltargets this population with equivalent efficiency but had no detrimental effects onCD34+CD38neg frequency. In summary, we demonstrate a high-frequency transductionprotocol which preserves the therapeutically relevant primitive subpopulation ofhuman hematopoietic cells.
C).Direct exposure of CD34+ target cells to VCM depletes the primitive CD34+CD38negsubpopulation by more than 30%, whereas the optimized VCM-free infection protocoltargets this population with equivalent efficiency but had no detrimental effects onCD34+CD38neg frequency. In summary, we demonstrate a high-frequency transductionprotocol which preserves the therapeutically relevant primitive subpopulation ofhuman hematopoietic cells.