US 12,213,466 B2
Process for using CRISPR to transfect primordial germ cells in avians
Scott Geoffrey Tyack, Grovedale (AU)
Assigned to COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION, Acton (AU); and AVIAGEN, Huntsville, AL (US)
Filed by Aviagen, Huntsville, AL (US); and Commonwealth Scientific and Industrial Research Organisation, Acton (AU)
Filed on Jun. 27, 2022, as Appl. No. 17/809,219.
Application 17/809,219 is a continuation of application No. 16/384,782, filed on Apr. 15, 2019, granted, now 11,369,096.
Application 16/384,782 is a continuation of application No. 14/394,712, granted, now 10,897,881, issued on Jan. 26, 2021, previously published as PCT/AU2013/000414, filed on Apr. 19, 2013.
Claims priority of provisional application 61/783,823, filed on Mar. 14, 2013.
Claims priority of provisional application 61/636,331, filed on Apr. 20, 2012.
Claims priority of application No. 2013204327 (AU), filed on Apr. 12, 2013.
Prior Publication US 2023/0189769 A1, Jun. 22, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. A01K 67/0275 (2024.01); A01K 67/0271 (2024.01); C12N 15/85 (2006.01); C12N 15/873 (2010.01); C12N 15/88 (2006.01); C12N 15/89 (2006.01); C12N 15/90 (2006.01); C12N 15/113 (2010.01); C12N 15/87 (2006.01)
CPC A01K 67/0275 (2013.01) [A01K 67/0271 (2013.01); C12N 15/8509 (2013.01); C12N 15/873 (2013.01); C12N 15/88 (2013.01); C12N 15/89 (2013.01); C12N 15/902 (2013.01); A01K 2217/058 (2013.01); A01K 2227/30 (2013.01); A01K 2267/02 (2013.01); C12N 15/1131 (2013.01); C12N 15/87 (2013.01); C12N 2517/02 (2013.01); C12N 2800/90 (2013.01)] 8 Claims
 
1. A method of incorporating exogenous DNA into the genome of chicken or quail primordial germ cells (PGCs), the method comprising: injecting a transfection mixture comprising:
a) a DNA polynucleotide encoding a guide RNA (gRNA) and a Cas9 nuclease;
b) exogenous DNA for integration into the genome of the PGCs; and
c) a cationic lipid
into a blood vessel of:
i) a stage 13-17 chicken embryo, or
ii) an equivalent stage quail embryo,
such that the exogenous DNA is integrated into the genome of the PGCs.