J Bacteriol Virol.  2007 Mar;37(1):31-38. 10.4167/jbv.2007.37.1.31.

Production of Recombinant Retroviruses Encoding Human Flt3 Ligand and IL-6 and Establishment of Genetically Modified OP9 Mouse Stromal Cells

• Kang HB
• Kim YE
• Kang HJ
• Lee AR
• Seok DE
• Lee Y

• KMID: 1513550
• DOI: http://doi.org/10.4167/jbv.2007.37.1.31

Abstract

Flt3 Ligand (FL) and IL-6 are multifunctional cytokines implicated in normal hematopoiesis and ex vivo expansion of hematopoietic stem cells. Retroviral vectors are useful for stable expression of genes in many cells. Here, we aimed to produce retroviral vectors directing expression of human FL and IL-6 genes. Recombinant retroviral vectors containing human genes for FL and IL-6 were constructed using a retroviral vector pLXSN. Recombinant retroviruses were produced from GP2-293 cells with the aid of pseudo-envelope protein gene VSV-G, and efficiently transduced to a mouse stromal cell line OP9. Genetically modified OP9 cells clearly showed expression of human FL or IL-6 gene at the mRNA level determined by RT-PCR. Based on the results from ELISA, human FL and IL-6 were detected in the cell culture medium of OP9/FL and OP9/IL-6 cells, respectively. As the recombinant human FL and IL-6 proteins are successfully produced and secreted to the culture medium, this system can be useful in future application such as ex vivo expansion of hematopoietic stem cells and differentiation of embryonic stem cells.

Keyword

Hematopoiesis; Retrovirus; Stromal cell; Flt3 Ligand; IL-6

MeSH Terms

Animals
Cell Culture Techniques
Cytokines
Embryonic Stem Cells
Enzyme-Linked Immunosorbent Assay
Hematopoiesis
Hematopoietic Stem Cells
Humans*
Interleukin-6*
Mice*
Retroviridae*
RNA, Messenger
Stromal Cells*
Zidovudine
Cytokines
Interleukin-6
RNA, Messenger
Zidovudine

Figure

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  Figure 1. Maps of the retroviral vectors used in this study. pLXSN including LTR promoter and Neo resistant marker was used as a parental cloning vector. Eco RI and Bam HI sites were used for cloning of FL and IL-6.

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  Figure 2. Expression of human FL and IL-6 genes in GP2-293 cells transfected with retroviral vectors. GP2–293 cells were transfected with retroviral vectors and selected in the presence of G418. Total RNAs were prepared from the G418-resistant cells and analyzed by RT-PCR. (A) Expression of human FL mRNA in GP2-293/FL cells. (B) Expression of human IL-6 mRNA in GP2-293/IL-6 cells. Expression of GAPDH was used as a RNA amount control.

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  Figure 3. Titration of retroviruses. Viral supernatants of the packaging cells GP2-293/FL and GP2–293/IL-6 were harvested and NIN3T3 cells were transduced with 10-fold serial dilutions of the retroviral supernatant (102 to 106 dilutions) or untransduced (mock). After two-week culture in the presence of G418, numbers of G418-resistant colonies were counted. (A) Representative result of titration experiments. (B) Calculated titers of the retroviruses.

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  Figure 4. Expression of human FL and IL-6 genes in OP9 cells transduced with retroviruses. OP9 cells were infected by retroviruses and selected in the presence of G418. Total RNAs were prepared from the G418-resistant cells and analyzed by RT-PCR. (A) Expression of human FL mRNA in OP9/FL cells. (B) Expression of human IL-6 mRNA in OP9/IL-6 cells. Expression of GAPDH was used as a RNA amount control.

• 

  Figure 5. Expression of human FL and IL-6 proteins in the culture media of OP9/FL and OP9/IL6 cells. Culture supernatants of OP9/control, OP9/FL and OP9/IL6 cells were harvested and the amounts of human FL (A) and IL-6 (B) proteins were measured by ELISA method. Data are presented as a mean value of triplicate experiments.

Reference

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