US 11,781,146 B2
Vector including a translation-impaired dihydrofolate reductase gene cassette and ubiquitously acting chromatin opening element
Yasuhiko Suzuki, Hokkaido (JP); Miki Nakagawa, Hokkaido (JP); Yayoi Kameda, Hokkaido (JP); Satoru Konnai, Hokkaido (JP); Tomohiro Okagawa, Hokkaido (JP); Naoya Maekawa, Hokkaido (JP); Shinya Goto, Hokkaido (JP); Yamato Sajiki, Hokkaido (JP); Kazuhiko Ohashi, Hokkaido (JP); Shiro Murata, Hokkaido (JP); Yuzuru Kitahara, Tokyo (JP); and Keiichi Yamamoto, Osaka (JP)
Assigned to NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY, Hokkaido (JP); and FUSO PHARMACEUTICAL INDUSTRIES, LTD., Osaka (JP)
Appl. No. 17/54,935
Filed by National University Corporation Hokkaido University, Hokkaido (JP); and Fuso Pharmaceutical Industries, Ltd., Osaka (JP)
PCT Filed May 13, 2019, PCT No. PCT/JP2019/018899
§ 371(c)(1), (2) Date Nov. 12, 2020,
PCT Pub. No. WO2019/225372, PCT Pub. Date Nov. 28, 2019.
Claims priority of application No. 2018-099704 (JP), filed on May 24, 2018; and application No. 2018-168591 (JP), filed on Sep. 10, 2018.
Prior Publication US 2021/0254079 A1, Aug. 19, 2021
Int. Cl. C12N 15/67 (2006.01); C12N 15/66 (2006.01); C12N 15/85 (2006.01)
CPC C12N 15/67 (2013.01) [C12N 15/66 (2013.01); C12N 15/85 (2013.01); C12Y 105/01003 (2013.01)] 20 Claims
 
1. An expression vector comprising the following (a), (b) and (c):
(a) a translation-impaired dihydrofolate reductase gene cassette (translation-impaired DHFR gene cassette) comprising a region with altered codons, wherein the altered codons comprise GCA for alanine, CGA for arginine, AAU for asparagine, GAU for aspartic acid, UGU for cysteine, CAA for glutamine, GAA for glutamic acid, GGU for glycine, CAU for histidine, UUA for leucine, AAA for lysine, CCA for proline, UUU for phenylalanine, UCA for serine, ACU for threonine, UAU for tyrosine, and/or GUA for valine, and wherein the region with altered codons accounts for 30% or more of the full length of the DHFR gene from the 5′ end of the DHFR gene;
(b) a gene cassette comprising a cloning site for integration of a foreign gene between a transcriptionally active promoter and a stable polyadenylation signal; and
(c) a ubiquitously acting chromatin opening element (UCOE),
wherein the UCOE comprises the nucleotide sequence as shown in SEQ ID NO: 1.