Iterative in vivo assembly of large and complex transgenes by combining the activities of phiC31 integrase and Cre recombinase.
Iterative in vivo assembly of large and complex transgenes by combining the activities of phiC31 integrase and Cre recombinase.
复制标题
通过将PHIC31积分酶和CRE重组酶的活性结合的大型和复杂转基因的体内组装。
DOI：
10.1093/nar/gni192
复制
发表时间：
2005-12-15
影响因子：
14.9
通讯作者：
Brown, WRA
中科院分区：
文献类型：
--
作者：
Dafhnis-Calas, F;Xu, ZY;Haines, S;Malla, SK;Smith, MCM;Brown, WRA
我们使用φC31整合酶将大的DNA序列导入脊椎动物基因组，并测量了完整DNA整合效率与插入物大小的关系。长度为110 kb和140 kb的插入可以分别以约25%和10%的效率集成。为了克服构建长度超过~ 150 kb的转基因的问题，我们建立了一种方法，我们称之为；“迭代站点特定集成”（ISSI）。ISSI结合了φC31整合酶和Cre重组酶的活性，实现了转基因DNA序列的迭代和序列整合。原则上，该过程可以重复任意次数，从而允许整合数百个千碱基对长的DNA束。在实践中，它可能受到在程序的每个步骤中检查积分准确性所需的时间的限制。我们描述了两个ISSI实验，在其中一个实验中，我们构建了一个复杂的150 kb大小的脊椎动物着丝粒序列阵列。ISSI的基本原理适用于所有生物的转基因。因此，ISSI可以促进转基因植物中许多不同基因编码的生物合成途径的重建，大型脊椎动物基因座的转基因组装和细菌中完整基因组的合成。
We have used the φC31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than ∼150 kb we have established a method that we call; ‘Iterative Site Specific Integration’ (ISSI). ISSI combines the activities of φC31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.
登录
查看更多内容
影响因子：
3.7
作者：
IZUMI, M;MIYAZAWA, H;HANAOKA, F
通讯作者：
HANAOKA, F
DOI：
10.1073/pnas.0931425100
发表时间：
2003-05-13
影响因子：
11.1
作者：
Lin, L;Liu, YG;Li, BJ
通讯作者：
Li, BJ
影响因子：
7.7
作者：
Sauer, Brian
通讯作者：
Sauer, Brian
DOI：
10.1073/pnas.95.10.5505
发表时间：
1998-05-12
影响因子：
11.1
作者：
Thorpe, HM;Smith, MCM
通讯作者：
Smith, MCM
影响因子：
14.9
作者：
Hejna, JA;Johnstone, PL;Moses, RE
通讯作者：
Moses, RE