Lyle Zimmerman group project:

Gynogenetic screens for chemically-induced mutations

We have conducted a pilot phenotypic screen for chemically-induced mutations, using gynogenesis to uncover recessive phenotypes in the progeny of heterozygous carriers. (Goda et al, 2006)

Gynogenesis

Gynogenesis

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Manipulating ploidy in X. tropicalis

In gynogenesis, first haploid embryos are created by in vitro fertilisation with UV-irradiated sperm. As completion of second meiosis normally occurs after fertilisation in amphibian eggs, haploids can be rescued to a viable diploid state by a simple cold treatment 5' post-fertilisation ('early cold shock') suppressing extrusion of the second polar body and leading to retention of both sets of sister chromatids.

Genome manipulations in X. tropicalis

Genome manipulations in X. tropicalis

Haploid embryos are generated by 'mock-fertilizing' eggs with UV-irradiated sperm (which cannot contribute genetically), and can survive a few days. Haploids can be rescued by suppressing formation of the polar body with pressure treatment shortly after fertilization. Such 'gynogenetic diploid' embryos are derived entirely from the maternal genome, and can be used to uncover recessive phenotypes in accelerated genetic screens.

Since the rescued genome is entirely maternally-derived, early cold shock screens can reveal recessive phenotypes in embryos produced by individual heterozygous females, greatly reducing husbandry space requirements and labor in addition to saving an extra generation time.

Using a screen based on morphological inspection of gynogenetic progeny of candidate carrier females, we have identified a large number of mutations in a variety of developmental processes, shown in the ENU phenotype database (Goda et al, 2006, Abu-Daya et al, 2009). Gynogenesis also provides useful strategies for rapid mapping of mutations to specific chromosomes (Khokha et al, 2009). We have successfully mapped and cloned a number of these mutations (Abu-Daya et al, 2009).

Please contact Lyle Zimmerman (lzimmer@nimr.mrc.ac.uk) if you are interested in participating in the analysis of these phenotypes.

Rapid mapping by bulk segregant analysis of gynogenetic embryos

Rapid mapping by bulk segregant analysis of gynogenetic embryos

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(A) A frog carrying a recessive mutation m on the N strain (white chromosomes) is crossed to a polymorphic IC strain (black chromosomes). (B) ‘Mapcross’ hybrid F2 carrier inherits one chromosome from each parent. (C) Pools of ~20 phenotypically mutant and wild type gynogenetic embryos are collected (mutant pool represented). Unlinked chromosomes show equal contribution from white N and black IC alleles (gray chromosomes) in both mutant and wild type pools. However, on the chromosome containing the mutation, the mutant pool is greatly enriched for the white N centromeric allele; the wild type pool may contain either the IC allele or both N and IC. Centromere linkage can often be detected over large genetic distances in gynogenetic embryos. (D) Silver-stained gel showing pools of mutant and wild type embryos scored with polymorphisms at the 10 X. tropicalis centomeres. Linkage is detected to chromosome 3.

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