Programming cells by multiplex genome engineering 多重基因组工程的程序设计.pptVIP

Multiplex Automated Genome Engineering (MAGE) for large-scale programming and evolution of cells. 报告人：陈艳 in vivo methods such as recombination-based genetic engineering (recombineering) have enabled efficient modification of single genetic targets using single-stranded DNA (ssDNA) no such attempts have been made to modify genomes on a large and parallel scale. MAGE 多基因工程(MAGE) MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. the process is cyclical and scalable MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). 单基因工程只能改良生物的单基因性状，不能改变高等生物的多基因性状 而多基因工程非但能改良生物品种而且能创造生物新物种 According to “High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides.” by Ellis, H. M et. from PNAS λ Red-mediated ssDNA recombination provides a rapid and highly efficient approach for generating recombinant DNA molecules. ------ssDNA Recombination Requires Only Beta. 与双链DNA重组机制不同: Red 重组系统由λ噬菌体 exo、 bet、 gam 三个基因分别编码的三种蛋白组成: Exo 蛋白是一种核酸外切酶 ,结合在双链DNA的末端 ,从5′端向3′端降解DNA ,产生 3′突出端; Beta 蛋白结合在单链DNA 上 ,介导互补单链DNA退火; Gam蛋白可与 RecBCD酶结合,抑制其降解外源DNA的活性。 Red同源重组技术具有同源序列短(40～60 bp) 、重组效率高的特点。 这种技术可在DNA靶标分子的任意位点进行基因敲除、敲入、点突变等操作 ,无需使用限制性内切酶和连接酶  λ Red-mediated ssDNA recombination provides a rapid and highly efficient approach for generating recombinant DNA molecules. Because this process is highly efficient, one can directly screen for mutations in the absence of selection.Oligo-mediated recombination occurs in both E. coli and yeast, suggesting that recombination via annealing of ssDNAs may occur in a wide range of organisms. Thus, recombineering with ssDNA may be applicable to higher eukaryotes as well, perhaps by a mechanism as simple as overexpressing Beta or a functionally