US 11,840,696 B2
Methods of introducing multiple expression constructs into a eukaryotic cell
Qiming Jin, Sacramento, CA (US); Jeffrey Shasky, Davis, CA (US); Donna Moyer, Davis, CA (US); Abigail Jang, Roseville, CA (US); Gloria Muzzi-Erichsen, Vacaville, CA (US); and Cara Kleindienst, Dixon, CA (US)
Assigned to Novozymes A/S, Bagsvaerd (DK)
Filed by Novozymes A/S, Bagsvaerd (DK)
Filed on Jul. 9, 2019, as Appl. No. 16/506,201.
Application 16/506,201 is a division of application No. 15/556,439, granted, now 10,385,352, previously published as PCT/US2016/021568, filed on Mar. 9, 2016.
Claims priority of provisional application 62/207,650, filed on Aug. 20, 2015.
Claims priority of provisional application 62/130,455, filed on Mar. 9, 2015.
Prior Publication US 2019/0330644 A1, Oct. 31, 2019
Int. Cl. C12N 15/79 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/79 (2013.01) [C12N 15/902 (2013.01); C12N 2800/30 (2013.01)] 17 Claims
 
1. A eukaryotic cell for expressing multiple heterologous proteins of interest, comprising:
(a) one or more first target loci each comprising a pair of a first recombination recognition site and a second recombination recognition site;
(b) one or more second target loci each comprising a first fragment of a first selectable marker lacking a selectable function;
(c) one or more first constructs, wherein the one or more first constructs each comprises one or more first expression cassettes each comprising a first polynucleotide encoding a first protein of interest, wherein in each of the first constructs the one or more first expression cassettes are flanked on one side by the first recombination recognition site and on the other side by the second recombination recognition site, wherein the first and second recombination recognition sites flanking the first expression cassettes are the same as the first and second recombination recognition sites of the first target loci; and
(d) one or more second constructs, wherein the one or more second constructs each comprises one or more second expression cassettes each comprising a second polynucleotide encoding a second protein of interest, wherein in each of the second constructs the one or more second expression cassettes are flanked on one side by a homologous region of the second target locus and on the other side by a second fragment of the first selectable marker that lacks the selectable function, wherein the second fragment comprises a sequence overlapping homologously a sequence of the first fragment of the first selectable marker of the second target loci.