Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs.
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http://dx.doi.org/10.1016/j.ydbio.2016.03.006 | DOI Listing |
Cold Spring Harb Protoc
December 2022
School of Natural Sciences, University of Central Missouri, Warrensburg, Missouri 64093, USA
We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing in embryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR-Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilized eggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F generation.
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June 2022
School of Natural Sciences, University of Central Missouri, Warrensburg, Missouri 64093, USA
In CRISPR-Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency.
View Article and Find Full Text PDFMethods Mol Biol
June 2019
Department of Biology, The University of Iowa, Iowa City, IA, USA.
The early development of Xenopus critically depends on maternal components stored in the egg. Because important events such as axis formation are triggered immediately after fertilization, it is often desirable to perturb gene function before this occurs. Oocytes can be experimentally manipulated in vitro, prior to maturation, and subsequently fertilized or otherwise activated to develop, and then observed for any embryological defects.
View Article and Find Full Text PDFMethods Mol Biol
May 2019
Cincinnati Children's Research Foundation, Cincinnati, OH, USA.
We have taken advantage of the well-established oocyte host transfer technique to optimize a method for CRISPR editing of Xenopus that provides an efficient non-mosaic targeted insertion of small DNA fragment through homology-directed repair mechanism.
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October 2018
Department of Biology, The University of Iowa, Iowa City, Iowa 52242-1324
This protocol details the oocyte host-transfer method in , using transplantation by intraperitoneal injection. This approach is suitable for the overexpression of mRNAs and for the use of antisense oligonucleotides to deplete maternal mRNAs, which are not replaced until zygotic genome activation in the mid-blastula transition. oocyte host-transfer can also be used for highly efficient mutagenesis in the F generation by prefertilization injection of genome editing reagents.
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