Show simple item record

dc.contributor.authorNottle, MB
dc.contributor.authorSalvaris, EJ
dc.contributor.authorFisicaro, N
dc.contributor.authorMcIlfatrick, S
dc.contributor.authorVassiliev, I
dc.contributor.authorHawthorne, WJ
dc.contributor.authorO'Connell, PJ
dc.contributor.authorBrady, JL
dc.contributor.authorLew, AM
dc.contributor.authorCowan, PJ
dc.date.accessioned2020-12-17T03:32:54Z
dc.date.available2020-12-17T03:32:54Z
dc.date.issued2017-08-16
dc.identifierpii: 10.1038/s41598-017-09030-6
dc.identifier.citationNottle, M. B., Salvaris, E. J., Fisicaro, N., McIlfatrick, S., Vassiliev, I., Hawthorne, W. J., O'Connell, P. J., Brady, J. L., Lew, A. M. & Cowan, P. J. (2017). Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9. SCIENTIFIC REPORTS, 7 (1), https://doi.org/10.1038/s41598-017-09030-6.
dc.identifier.issn2045-2322
dc.identifier.urihttp://hdl.handle.net/11343/254930
dc.description.abstractXenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.
dc.languageEnglish
dc.publisherNATURE PUBLISHING GROUP
dc.rights.urihttps://creativecommons.org/licenses/by/4.0
dc.titleTargeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9
dc.typeJournal Article
dc.identifier.doi10.1038/s41598-017-09030-6
melbourne.affiliation.departmentMedical Biology (W.E.H.I.)
melbourne.affiliation.departmentMedicine (St Vincent's)
melbourne.affiliation.facultyMedicine, Dentistry & Health Sciences
melbourne.source.titleScientific Reports
melbourne.source.volume7
melbourne.source.issue1
melbourne.identifier.nhmrc1061868
dc.rights.licenseCC BY
melbourne.elementsid1229593
melbourne.contributor.authorLew, Andrew
melbourne.contributor.authorSalvaris, Evelyn
melbourne.contributor.authorCowan, Peter
melbourne.contributor.authorBrady, Jamie
dc.identifier.eissn2045-2322
melbourne.identifier.fundernameidNHMRC, 1061868
melbourne.accessrightsOpen Access


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record