Homologous recombination in Xenopus and Zebrafish

描述(申请人提供)：这些研究的长期目标是为脊椎动物胚胎的基因操作产生新的方法，最终目的是了解基因在发育过程中的功能。研究人员提议进行实验，以验证噬菌体蛋白对可以刺激脊椎动物非洲爪哇和斑马鱼胚胎中的同源重组(HR)的假设。噬菌体蛋白对包括来自lambda噬菌体的Red_和Red__，以及来自lambdoid噬菌体Rac的Ress和Rect。这些蛋白质对已被用于利用短同源区域进行有效的基因转化和缺口修复。重组涉及5‘-3’外切酶(Ress或Red-α)与单链结合蛋白(RECT或Red-Beta)之间的合作。我们的合作者A.F.斯图尔特率先使用这些蛋白质在大肠杆菌中启动同源重组。最近，斯图尔特博士的实验室已经证明，这些蛋白质可以通过单链寡核苷酸靶向特定序列，促进小鼠ES细胞中的同源重组。这被称为单链寡核苷酸修复的单链寡核苷酸修复。这项探索性拨款将研究非洲爪哇或斑马鱼早期胚胎中的几种方法，以利用噬菌体蛋白对促进SSOR来靶向特定的染色体序列。研究人员建议：(1)建立非洲爪哇卵胞浆抽提物中SSOR的检测方法，以探索非洲爪哇胚胎细胞质中对HR的特殊要求。该检测将检查染色体外质粒上抗生素耐药基因修复的要求，并将使用大肠杆菌菌落读数。(2)研究人员将针对热带非洲爪哇胚胎中的染色体绿色荧光蛋白(GFP)转基因，以产生视觉上可筛选的功能丧失表型。纯化的噬菌体蛋白和单链寡核苷酸将被用于靶向GFP转基因，通过编码改变、移码突变或停止密码子插入来改变GFP表达。(3)研究人员将探索利用SSOR来定位几个斑马鱼染色体基因座的条件，以产生具有良好特征的点突变，从而提供可评分的表型。

DESCRIPTION (provided by applicant): The long-term objective of these studies is to generate novel methodologies for genetic manipulation of vertebrate embryos for the ultimate purpose of understanding gene function during development. The investigators propose experiments to test the hypothesis that bacteriophage protein pairs can stimulate homologous recombination (HR) in vertebrate Xenopus and Zebrafish embryos. The bacteriophage protein pairs include Red__ and Red__ from phage lambda, and RecE and RecT from the lambdoid prophage Rac. The protein pairs have been employed for both efficient gene conversion and gap repair using short homology regions. Recombination involves co-operation between a 5'-3' exonuclease (RecE or Red-alpha with a single strand binding protein (RecT or Red-Beta). Our collaborator, A.F. Stewart, pioneered the use of these proteins to initiate homologous recombination in E. coli. Recently Dr. Stewart's lab has demonstrated that these proteins can promote homologous recombination in mouse ES cells using single stranded oligonucleotides to target specific sequences. This has been termed ssOR for single-strand oligo repair. This exploratory grant will investigate several approaches in early Xenopus or Zebrafish embryos to target specific chromosomal sequences using the phage protein pairs to promote ssOR. The investigators propose to (1) develop assays for ssOR in cytoplasmic extracts of Xenopus eggs to explore the specific requirements for HR in the Xenopus embryonic cytoplasm. The assay will examine requirements for repair of antibiotic resistance genes on extrachromosomal plasmids and will use an E.coli colony readout. (2) The investigators will target a chromosomal Green Fluorescent Protein (GFP) transgene in Xenopus tropicalis embryos to generate visually screenable loss of function phenotypes. Purified phage proteins and single strand oligos will be used target a GFP transgene to alter the expression of GFP expression through coding changes, frameshift mutation, or stop codon insertion. (3) The investigators will explore conditions to target several zebrafish chromosomal loci using ssOR to generate well-characterized point mutations that give scorable phenotypes.