Recombination-mediated genetic engineering of Plasmodium berghei DNA.
Recombination-mediated genetic engineering of Plasmodium berghei DNA.
复制标题
伯氏疟原虫 DNA 重组介导的基因工程。
DOI：
10.1007/978-1-62703-026-7_8
复制
发表时间：
2013
影响因子：
--
通讯作者：
O. Billker
中科院分区：
文献类型：
--
作者：
Claudia Pfander;B. Anar;M. Brochet;J. Rayner;O. Billker
由于伯氏疟原虫的腺嘌呤和胸腺嘧啶（AT）核苷酸含量高，用传统的限制性和结扎方法难以在大肠杆菌中对其DNA进行处理。这限制了我们克隆大基因和生成复杂载体来修饰寄生虫基因组的能力。我们在这里描述了一种使用lambda Red重组酶修饰在线性、低拷贝、噬菌体衍生载体中构建的柏氏假体基因组DNA文库插入的方案。该方法使用的引物长度为50 bp，为抗生素耐药性标记提供了足够的同源性，可以有效地与大肠杆菌中的伯氏杆菌基因组DNA插入物重组。在随后的体外Gateway反应中，将细菌标记物替换为用于伯氏假体选择的盒式培养基。然后释放插入物并用于转染。我们在这里描述的基本技术可以用于产生高效的载体，用于基因删除、标记、靶向诱变或更大基因组区域的遗传互补。
DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.