BACKGROUND A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. RESULTS Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. CONCLUSIONS An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.

中文翻译：

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包装伪型整合酶缺陷型慢病毒的优化协议。

背景技术已经开发了许多整合酶缺陷型慢病毒（IDLV）包装系统，以产生整合缺陷型慢病毒，用于基因递送和染色体表达。然而，尽管它们的需求不断增长，但尚未进行比较研究来系统地评估不同突变体在病毒包装和基因表达上的表现效率。结果定点诱变用于非整合型慢病毒包装（NILVP）产生五个整合酶缺陷型突变体。然后将五个突变体分别掺入以形成不同的整合酶缺陷型慢病毒质粒包装混合物，同时保持其他包装因子不变。将来自EF1启动子的表达慢病毒的CD511B-1与五种不同慢病毒包装混合物中的每一种包装在一起，制成假型病毒颗粒。基于GFP在HT1080细胞中的表达来评估每个整合酶缺陷型突变体的性能和包装效率，而野生型慢病毒包装混合物用作对照。在五种整合酶突变候选物中，选择了具有最高GFP转基因表达水平的一种进行进一步表征。通过定量聚合酶链反应证实了该候选物的非整合性质，并使用分裂和非分裂细胞对其进行了表征。最后，开发了使用该整合酶缺陷突变体的NILVP的详细标准方案。结论建立了一种有效的慢病毒包装系统，用于生产整合型慢病毒。该系统与大多数现有的慢向量兼容，可用于转导分裂细胞和非分裂细胞。