Development of a method for efficient transfer and expression of genes in hematopoietic cells.

开发一种在造血细胞中有效转移和表达基因的方法。

• 批准号: 10557091
• 负责人: ITOU Katsuhiko
• 金额: $ 7.49万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557091/
• 关键词: hematopoietic cell; retroviral vector; stroma; レトロウィルス・ベクター; レトロウイルス; 造血幹細胞; シュードタイプ・ウイルス; GALV; VSV G蛋白

A novel murine stromal cell line ,HESS-M28, was established, which supports the expansion of human CD34+CD38-cells more than 300-fold in vitro in the presence of human IL-3 and SCF. Utilizing this cells, Attempt was made to evaluate cis-acting elements of retroviral vectors in human primitive hematopoietic cells.Cord blood cells were cultured on top of the mixed cell layers of the stromal cell line, HESS-M28, and retroviral vector producing cells. The FMEV-type vector ,SF/Lyt, contained the spleen focus-forming virus U3 and the MESV primer binding site (PBS), while MO3/Lyt contained the U3 region and PBS from MoMLV. Following transduction by the FMEV-type and the MoMLV-based vectors, expression of the marker gene, murine CD8(mCD8), was examined in CD34-, CD34+ and CD34+CD38-cells. In CD34+and CD34+CD38-cells, expression of mCD8 was higher with the FMEV-type vector, SF/Lyt, compared to the cells transduced by the MoMLV-based vector,MO3/Lyt,although the expression was comparable in CD34-cells. Expression of marker genes was also confirmedimed in long-term culture-initiating cells(LTC-ICs)and SCID-repopulating cells(SRCs).

建立了一种新型小鼠基质细胞系 HESS-M28，在人 IL-3 和 SCF 存在的情况下，该细胞系支持人 CD34+CD38 细胞在体外扩增 300 倍以上。利用该细胞，尝试评估人类原始造血细胞中逆转录病毒载体的顺式作用元件。在基质细胞系、HESS-M28和逆转录病毒载体产生细胞的混合细胞层上培养脐带血细胞。 FMEV 型载体 SF/Lyt 含有脾病灶形成病毒 U3 和 MESV 引物结合位点 (PBS)，而 MO3/Lyt 含有 MoMLV 的 U3 区域和 PBS。通过 FMEV 型和基于 MoMLV 的载体转导后，在 CD34-、CD34+ 和 CD34+CD38-细胞中检查标记基因鼠 CD8(mCD8) 的表达。在 CD34+ 和 CD34+CD38 细胞中，与基于 MoMLV 的载体 MO3/Lyt 转导的细胞相比，FMEV 型载体 SF/Lyt 的 mCD8 表达较高，尽管在 CD34 细胞中的表达相当。标记基因的表达也在长期培养起始细胞(LTC-ICs)和SCID再生细胞(SRCs)中得到证实。

项目成果

Tsuji T et al.: "Exvivo generation of CD34(+)cells from CD34(-) hematopoietic cells"Blood. 94. 4053-4059 (1999)

Tsuji T 等人：“从 CD34(-) 造血细胞离体生成 CD34(-) 细胞”血液。

Tsuji T, ltoh K, Nishimura-Morita Y, Watanabe Y, Hirano D, Mori KJ and Yatsunami K.: "CD34high+CD38low/- cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation."Leukemia. 13. 1409-1419 (199

Tsuji T、ltoh K、Nishimura-Morita Y、Watanabe Y、Hirano D、Mori KJ 和 Yatsunami K.：“异种共培养系统中产生的 CD34high CD38low/- 细胞能够进行长期造血和多重分化。”白血病

Nishiyama H et al.: "Decreased expression of cold-induced RNA-binding protein(CIRP)in male germ cells at elerated temperature" Am.J.Path.152. 289-296 (1998)

Nishiyama H 等人：“升高温度下雄性生殖细胞中冷诱导 RNA 结合蛋白 (CIRP) 的表达降低”Am.J.Path.152。

Tsuji T et al.: "Vascular smooth muscle differentiation of murine stroma : a sequential model"Exp. Hematol.. 27. 1782-1795 (1999)

Tsuji T 等人：“小鼠基质的血管平滑肌分化：顺序模型”实验。

Itoh K et al.: "Retroviral vector-mediated gene expression in human CD34+CD38cells expanded in vitro : cis-elements of FMEV are superior to those of *"Hum. Gene Ther.. 11. 271-284 (2000)

Itoh K 等人：“体外扩增的人 CD34 CD38 细胞中逆转录病毒载体介导的基因表达：FMEV 的顺式元件优于*”Hum。