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Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

ABSTRACT: Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication of defensin7 with sequence identity at the junction, suggesting nonallelic homologous recombination between defensin7 and defensin6 The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression of defensin7 was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins.

SUBMITTER: Lee MO

PROVIDER: S-EPMC5137700 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

Lee Mi Ok MO Bornelöv Susanne S Andersson Leif L Lamont Susan J SJ Chen Junfeng J Womack James E JE

Proceedings of the National Academy of Sciences of the United States of America 20161114 48

Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the ...[more]

PMID: 27849592

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Project description:Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Dataset Information

Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

Publications

Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

Similar Datasets

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