Development of a novel retrovirus vector system for hematopoietic Stem cells

该项目的目标如下：1。评价由SFFV β LTR和MESV前导序列组成的新型逆转录病毒载体的基因表达.基于基质细胞的新型包装细胞的建立及评价.以SFFV p为基础构建的含VSV G蛋白的假型逆转录病毒载体的评价。构建了一系列逆转录病毒载体。使用基于NIH/3 T3的包装细胞，分离这些载体产生细胞。在造血祖细胞中比较这些载体的表达水平。其中，FMEV型载体中的标记基因表达量最高，该载体含有SFFVp LTR和MESV前导序列.将携带gal/pol序列的表达载体和携带MoMLV env序列的表达载体的质粒DNA引入基质细胞系MS-5中。发现这些新型的基于基质细胞的包装细胞不稳定，不能产生高滴度的病毒载体.基于SFFVp的逆转录病毒载体可以用VSV G蛋白假型化，并且这些颗粒成功地转导细胞。

The aims of the project was as follows ;1. Evaluation of the gene expression by a novel retroviral vector which consisted of the SFFVp LTR and the MESV leader sequences.2. Establishment and the evaluation of novel packaging cells based on stromal cells.3. Evaluation of the pseudo-typed retroviral vectors based on SFFVp with VSV G-protein.We have obtained the following results ;1. A series of retroviral vectors were constructed. Using the packaging cells based on NIH/3T3, these vector-producing cells were isolated. Expression levels of these vectors were compared in hematopoietic progenitors. The highest expression of a marker gene among these was obtained by the FMEV-type vector, which contained the SFFVp LTR and the MESV leader sequences.2. Plasmid DNAs of the expression vector carrying gal/pol sequences and that carrying the MoMLV env sequences were introduced into a stromal cell line, MS-5. These novel stromal cell-based packaging cells were found to be unstable to produce high titer of viral vectors.3. SFFVp based retroviral vectors could be pseudo-typed with VSV G-protein and these particles successfully transduced the cells.