Idioma:

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

Crispo, M ; Mulet, A P ; Tesson, L ; Barrera, N ; Cuadro, F ; dos Santos-Neto, P C ; Nguyen, T H ; Crénéguy, A ; Brusselle, L ; Anegón, I ; Menchaca, A Veitia, Reiner Albert

PloS one, 2015-08, Vol.10 (8), p.e0136690-e0136690 [Periódico revisado por pares]

United States: Public Library of Science

Texto completo disponível

Citações Citado por

Enviar para

Título:
Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes
Autor: Crispo, M ; Mulet, A P ; Tesson, L ; Barrera, N ; Cuadro, F ; dos Santos-Neto, P C ; Nguyen, T H ; Crénéguy, A ; Brusselle, L ; Anegón, I ; Menchaca, A
Veitia, Reiner Albert
Assuntos: Analysis ; Animal behavior ; Animal genetic engineering ; Animal models ; Animals ; Animals, Genetically Modified ; Blastocysts ; Body weight ; Codons ; CRISPR ; CRISPR-Cas Systems ; Cytoplasm ; Deoxyribonucleic acid ; DNA ; DNA methylation ; DNA sequencing ; Efficiency ; Embryonic development ; Embryos ; Female ; Females ; Gene Knockout Techniques ; Gene sequencing ; Genetic modification ; Genetically modified animals ; Genomes ; Human health and pathology ; Immunology ; Laboratory animals ; Life Sciences ; Livestock ; Microinjection ; Microinjections ; MSTN gene ; Muscles ; Muscular dystrophy ; Mutation ; Myostatin ; Myostatin - genetics ; Neonates ; Nucleotide sequencing ; Ovis aries ; Pregnancy ; Sheep ; Sheep, Domestic - genetics ; Technology ; Wool ; Zygote ; Zygotes
É parte de: PloS one, 2015-08, Vol.10 (8), p.e0136690-e0136690
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Conceived and designed the experiments: MC IA AM. Performed the experiments: MC APM NB FC PCdSN TN IA AM. Analyzed the data: MC APM LT TN AC LB IA AM. Contributed reagents/materials/analysis tools: MC IA AM. Wrote the paper: MC APM TN IA AM.
Competing Interests: The authors have declared that no competing interests exist.
Descrição: While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.
Editor: United States: Public Library of Science
Idioma: Inglês

Links

Voltar para lista de resultados

Realização: Logos de Redes Sociais:

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

Crispo, M ; Mulet, A P ; Tesson, L ; Barrera, N ; Cuadro, F ; dos Santos-Neto, P C ; Nguyen, T H ; Crénéguy, A ; Brusselle, L ; Anegón, I ; Menchaca, A Veitia, Reiner Albert

United States: Public Library of Science

Buscando em bases de dados remotas. Favor aguardar.