Homologous recombination in Xenopus and Zebrafish

描述（由申请人提供）：这些研究的长期目标是产生脊椎动物胚胎遗传操作的新方法，最终目的是了解发育过程中的基因功能。研究人员提出实验来检验噬菌体蛋白对可以刺激脊椎动物非洲爪蟾和斑马鱼胚胎中的同源重组（HR）的假设。噬菌体蛋白质对包括来自λ噬菌体的Red__和Red__，以及来自拟杆状病毒原噬菌体Rac的RecE和RecT。蛋白质对已被用于有效的基因转换和缺口修复，使用短的同源区域。扩增涉及5 '-3'核酸外切酶（RecE或Red-alpha）与单链结合蛋白（RecT或Red-beta）之间的合作。我们的合作者A.F. Stewart等人率先使用这些蛋白质在大肠杆菌中启动同源重组。杆菌最近，Stewart博士的实验室已经证明，这些蛋白质可以促进小鼠ES细胞中的同源重组，使用单链寡核苷酸靶向特定序列。这被称为单链寡核苷酸修复的ssOR。这项探索性资助将研究早期非洲爪蟾或斑马鱼胚胎中的几种方法，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）开发非洲爪蟾卵细胞质提取物中ssOR的测定方法，以探索非洲爪蟾胚胎细胞质中HR的特定要求。该试验将检查染色体外质粒上抗生素耐药基因的修复要求，并将使用大肠杆菌菌落读数。(2)研究人员将在热带爪蟾胚胎中靶向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用于靶向GFP转基因，以通过编码改变、移码突变或终止密码子插入来改变GFP表达的表达。(3)研究人员将探索使用ssOR靶向几个斑马鱼染色体位点的条件，以产生具有良好特征的点突变，从而获得可评分的表型。

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.