US 11,732,273 B2
Methods and compositions for in situ germline genome engineering
Channabasavaiah Gurumurthy, Omaha, NE (US); Masato Ohtsuka, Kanagawa (JP); and Masahiro Sato, Kagoshima (JP)
Assigned to Board of Regents of the University of Nebraska, Lincoln, NE (US); and Tokai University Educational System, Tokyo (JP)
Filed by BOARD OF REGENTS OF THE UNIVERSITY OF NEBRASKA, Lincoln, NE (US); and TOKAI UNIVERSITY EDUCATIONAL SYSTEM, Tokyo (JP)
Filed on Feb. 24, 2020, as Appl. No. 16/799,398.
Application 16/799,398 is a continuation in part of application No. PCT/US2018/047748, filed on Aug. 23, 2018.
Claims priority of provisional application 62/549,644, filed on Aug. 24, 2017.
Prior Publication US 2020/0263198 A1, Aug. 20, 2020
Int. Cl. C12N 5/00 (2006.01); C12N 15/85 (2006.01); C12N 5/071 (2010.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); A01K 67/027 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/85 (2013.01) [A01K 67/0275 (2013.01); C12N 5/0608 (2013.01); C12N 5/0682 (2013.01); C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/907 (2013.01); A01K 2227/105 (2013.01); A01K 2267/03 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 11 Claims
 
1. A method for germline genome engineering in a subject having a reproductive organ containing a fertilized zygote comprising:
(a) isolating or obtaining the reproductive organ from the subject after a time period of about 12 hours to about 16.8 hours following insemination of the subject;
(b) introducing a reagent composition into the reproductive organ, the reagent composition comprising a nuclease system and/or an exogeneous polynucleotide; and
(c) electroporating the reproductive organ, wherein during electroporation, the reproductive organ contains the fertilized zygote,
wherein the reproductive organ is electroporated using an electroporator using the following parameters selected from the group consisting of:
A) poring pulse: 50 V, 5-ms pulse, 50-ms pulse interval, 3 pulse, 10% decay (±pulse orientation) and transfer pulse: 10 V, 50-ms pulse, 50-ms pulse interval, 3 pulse, 40% decay(+pulse orientation) and
B) square (mA), (+/−), Pd V: 60 V or 80 V, Pd A: 150 mA, Pd on: 5.00 ms, Pd off: 50 ms, Pd N: 3, Decay: 10%, Decay Type: Log.