Therapeutic agent for disease with apoptotic degeneration in eye tissue cell containing PEDF and FGF2
A drug and fibroblast technology, which is used in the field of therapeutic drugs for diseases associated with apoptosis and degeneration of ocular tissue cells containing PEDF and FGF2
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Embodiment 1
[0098] Example 1 Construction of VSV-G pseudotyped SIV vector
[0099] Vectors were constructed using the figure 1 The 4 plasmids (gene transfer vector, packaging vector, rev expression vector, VSV-G expression vector) shown in . Regarding the three types of gene transfer vectors, packaging vectors, and rev expression vectors, they were prepared by transforming the original vector plasmid (PCT / JP00 / 03955). As for the VSV-G expression vector, an unmodified original vector was used.
[0100] For plasmid preparation, various commercially available kits were used. Products from New England Biolabs were used as restriction enzymes, and QIAGEN kits (QIAquick PCR purification kit, QIAquick NucleotideRemoval kit, QIAquick Gel extraction kit, Plasmid Maxi kit) were used for plasmid DNA extraction, purification, and recovery. For PCR, TaKaRa's EX Taq enzyme was used, and the primers used were synthesized by an outsourced manufacturer (SIGMA GENOSYS JAPAN). Dephosphorylation of DNA t...
Embodiment 2
[0118] Example 2 Functional evaluation of the SIV vector carrying cPPT and WPRE
[0119] In order to investigate the introduction effect of cPPT and WPRE, in addition to carrying cPPT and WPRE at the same time, a vector carrying cPPT alone and WPRE alone was also produced, and compared with the original control. All gene transfer vectors used carried EGFP. The original type (sequence number: 33) was used as the packaging carrier.
[0120] 2-1. Preparation of SIV vector
[0121] The cell line 293T cells from human fetal kidney cells were divided into about 1×10 per 15cm plastic culture dish 7 Inoculate (70-80% density on the next day) and culture in 20 ml of D-MEM medium (Gibco BRL) containing 10% fetal bovine serum for 24 hours. After 24 hours of culture, the medium was replaced with 10 ml of OPTI-MEM medium (Gibco BRL) and used as transfected cells for later use.
[0122]Dissolve 6 μg of gene transfer vector, 3 μg of packaging vector, and 1 μg of VSV-G expression vector i...
Embodiment 3
[0134] Example 3 Mass preparation and concentration of SIV vectors carrying therapeutic genes
[0135] Such as figure 1 As shown, the SIV vector was prepared as follows on the basis of four kinds of plasmids: a modified gene transfer vector, a packaging vector, a rev expression vector, and a VSV-G expression vector. The vectors carrying the therapeutic genes of PEDF and FGF2 are produced in units of 20 15cm dishes.
[0136] According to each 15cm plastic Petri dish about 1×10 7 The 293T cells were inoculated (at a density of 70-80% on the next day), and cultured in 20 ml of D-MEM medium containing 10% fetal bovine serum for 24 hours. After 24 hours of cultivation, the medium was replaced with 10 ml of OPTI-MEM medium for transfection. Dissolve 10 μg of gene transfer vector, 5 μg of packaging vector, 2 μg of rev expression vector, and 2 μg of VSV-G expression vector in 1.5 ml of OPTI-MEM medium for each culture dish, then add 40 μl of PLUS Reagent reagent (Invitrogen) for st...
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Abstract
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Application Information
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