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US 12,735,719 B2Granted
Non-viral transgenesis
AssigneeThe Regents of the University of California
InventorsChih-Wen Ni, Chang-Ying Chiang
Life of this patent
Estimating the term…
Abstract
Provided herein are new compositions and methods for use in introducing transgenes into cells. The compositions are non-viral but achieve levels of transgene integration comparable to those obtained with viral-mediated methods, and can be used for targeted integration of a transgene at a specific genomic locus.
Claims12
A polynucleotide comprising:
- (a) one or more selection markers, wherein the selection markers are flanked by
- (b) first and second att sites, wherein the att sites are flanked by
- (c) first and second truncated retroviral long terminal repeats (LTRs), wherein the first truncated LTR is upstream of the first att site, the second truncated LTR is downstream of the second att site, and the first and second truncated retroviral LTRs are flanked by
- (d) recognition sites for a restriction enzyme, wherein cleavage of the recognition sites generates blunt ends;
- and
- (e) first and second 5′-ACTG-3′ sequences, present at or near the termini of the polynucleotide, wherein the second truncated LTR sequence comprises:
- (SEQ. ID NO. 5)
- TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGT
- ACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTA
- ACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC
- AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
- AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA.
9 more elements
A polynucleotide comprising:
- (a) one or more selection markers, wherein the selection markers are flanked by
- (b) first and second att sites, wherein the att sites are flanked by
- (c) first and second truncated retroviral long terminal repeats (LTRs), wherein the first truncated LTR is upstream of the first att site, the second truncated LTR is downstream of the second att site, and the first and second truncated retroviral LTRs are flanked by
- (d) recognition sites for a restriction enzyme, wherein cleavage of the recognition sites generates blunt ends;
- and
- (e) first and second 5′-ACTG-3′ sequences, present at or near the termini of the polynucleotide, wherein the second truncated LTR sequence comprises:
- TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACT GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGT GTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAG TCAGTGTGGAAAATCTCTAGCA (SEQ. ID NO. 5), and wherein the polynucleotide further comprises a transgene disposed between the first and second truncated retroviral LTRs.
4 more elements
Dependencies are inferred from the claim text.