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.
