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Laboratory Protocols & SOPs Library

Simian vacuolar virus type 1 vector

Published · Updated
Summary

Vacuolar viruses (FV) are non-pathogenic retroviruses that efficiently and safely transfer genes to different types of cells in different species. These viruses have a unique replication mechanism that is different from that of other retroviruses, which is an advantage of vacuolar viruses in mediating gene transfer. Their reverse transcription occurs late in the viral life cycle. At this point, at least 20% of the virus particles contain infectious double-stranded DNA genomes Author: T. Friedman et al.

Operation method

Preparation of SFV-1 Vector

Move

Preparation of SFV-1 Vector Material

reagents
CaCl2 (2m o l /L )

Chlorine (50m m o l /L)

Complete DMEM medium with 10 % FBS, 2 m m o l / L L-glutamine, l O O U / m l penicillin,

100ug/ml streptomycin

Fetal Bovine Serum (F B S )

2XHEPES buffer (HBS; 50 mmol/L HEPES, O.78 mmol/L Na2 HPO4, and 273 mmol/L NaCl)

H a n k balanced salt solution (H B S S )

Human embryonic kidney fibroblast-derived 293T cells
每 隔 3〜 4山 将 细 胞 以 1:5传 代 。 293丁细胞从八丁0：获 得 （0 ^ 1 5 7 3 ) 。 仪器 Apollo 离心浓缩棒（70k D a ; Orbital Bioscience， Topsfield， Massachusetts) 培养瓶' ( 25Cm2 和 75cm2) 滤 器 （(X45lLim，无菌包装，低蛋白质结合力， Millex-H V , Millipore) 倒置荧光显微镜 注 射 器 （IOml) 组织培养板（6 孔 和 24孔） 离 心 管 （conical, 15ml 和 50ml) 离 心 管 （1.5m l ，螺丝帽； Sarstedt) 方法____________ ___________________ ___________________________________ 用磷酸钙沉淀法转染DNA 1•转染前一天，每个75c m 2培养瓶接种5X 106个细胞。 转染当天，细胞密度应达到8 0 % 汇合。 2•用 2_634m l (263知1) H zO 稀释 12f z g S F V - l 载体、 12M g p C I g a g 、 6吨 pCIpol、 2吨 p C I e n v 0 用 超 纯 水 （autoclaved， Millipore)。 3. 加 366M12m o l / L CaCl2至 D N A 混合物并混勻。力 n 3m l 2 X H B S 到 D N A -CaCl2混合 液 ，室温放置lOinin。 4. 制 备 25p m o l /L 氯喹溶液：加 4M1 50m m o l /L 氯喹到6m l 完 全 D M E M 培养基中。用 6m l 这种氯喹溶液替换培养基。 ;•用枪头混合D N A -CaCl2- H B S 溶液，并产生I O s 的气泡，然后立即加到细胞中。 •孵育6〜8h 后 ，弃掉培养基，用 含 l O m m o l / L 丁酸钠的完全培养基代替。 丁酸钠处理不应超过24h , 丁酸钠能增加载体产量（ TanakaetaL 1991)。 ，毒载体的收集和浓缩 第二天早晨，用 6. 5m l 含 3m m o l / L 丁酸钠的新鲜完全培养基替换培养基。
至 少 80%的细胞应表达G F P , 可以直接从一个倒置荧光显微镜观察到。通 常 在 转 染4〜5d 后收集上清。 8.确定载体滴度：感染新鲜的293T 细胞，计 算 G F P 阳性细胞。 通 常 ，能 产 生 IO7个/m l的 SFV-I 载体颗粒。载体能在4 C 保 存 2 周而不会有明显损失。冻 融一次会导致载体浓度丢失10倍 。 9•为了增加转导效率，用离心柱将S F V -I 上清浓缩100倍 。将病毒上清以4000g 离心 2〇 m in，除掉细胞碎片，用 0.45M m 的滤膜过滤。 10.将过滤后的上清加到70k D a 的 A p o 丨 Io离心柱，室温离心30m i n 。病毒可在4°C 短期 保存，或一80°C 长期保存


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Cite this article

Aladdin Scientific. "Simian vacuolar virus type 1 vector" Aladdin Knowledge Base, updated 09/06/2025. https://www.aladdinsci.com/eu_pt/faqs/simian-vacuolar-virus-type-1-vector-en.html
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