Generation of cell lines to complement adenovirus vectors using recombination-mediated cassette exchange.
Generation of cell lines to complement adenovirus vectors using recombination-mediated cassette exchange.
复制标题
使用重组介导的盒式交换生成细胞系以补充腺病毒载体。
DOI：
10.1186/1472-6750-10-92
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发表时间：
2010-12-23
影响因子：
3.5
通讯作者：
Leppard KN
中科院分区：
文献类型：
--
作者：
Morris SJ;Farley DC;Leppard KN
腺病毒血清型 5 (Ad5) 具有许多有利于开发为基因治疗载体的特征。然而，当前 Ad5 载体的实用性受到瞬时转基因表达、毒性和免疫原性的限制。最有前途的载体形式是高容量型，它删除了所有病毒基因。然而，这些载体只能以相对较低的滴度并在辅助病毒的帮助下生产。因此，持续的挑战是生成更有效且仍可生长至高滴度的 Ad5 载体。我们的方法是生成补充细胞系，以支持具有新型晚期基因缺陷的 Ad5 载体的生长。我们使用 LoxP/Cre 重组介导的盒交换 (RMCE) 来生成表达由基因组 L4 区域编码的 Ad5 蛋白的细胞系，其产物在 Ad5 结构蛋白的表达中发挥关键作用。生成了一组 LoxP 亲代 293 细胞系，每个细胞系都包含一个 GFP 表达盒，该表达盒受插入随机基因组位置的四环素调节启动子的控制；该盒还在启动子和 GFP 序列之间包含一个 LoxP 位点。克隆表现出多种调节模式、稳定性和 GFP 表达水平。由于其 GFP 表达的严格诱导性和稳定性，克隆 A1 被确定为创建诱导细胞系的合适亲本。使用 LoxP 靶向、Cre 重组酶介导的 L4 盒插入来取代该亲本克隆中受调节启动子中的 GFP，生成细胞系 A1-L4。该细胞系表达 L4 100K、22K 和 33K 蛋白，其水平足以补充 L4-33K 突变体和 L4 缺失病毒。 RMCE 提供了一种从预先选择的亲本细胞系中快速生成 Ad5 互补细胞系的方法，该细胞系是根据其所需的转基因表达特征而选择的。可以选择亲本细胞系以进行高或低基因表达以及严格调节，从而使病毒蛋白表达能够反映感染过程中发现的情况。来自单亲本的细胞系将允许评估不同载体的生长，而不会出现不同的互补蛋白表达的复杂化。
Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.
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影响因子：
5
作者：
Kito, M;Itami, S;Shibui, T
通讯作者：
Shibui, T
影响因子：
64.5
作者：
CEPKO, CL;SHARP, PA
通讯作者：
SHARP, PA
影响因子：
5.4
作者：
Dedieu, JF;Vigne, E;Yeh, P
通讯作者：
Yeh, P
影响因子：
3.8
作者：
GRAHAM, FL;SMILEY, J;NAIRN, R
通讯作者：
NAIRN, R
影响因子：
4.8
作者：
HOWE, JR;SKRYABIN, BV;SCHMAUSS, C
通讯作者：
SCHMAUSS, C