Methods and compositions for targeted integration

摘要

Disclosed herein are methods and compositions for targeted integration of one or more copies of a sequence of interest using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain and integrase defective lentiviral donor constructs.

主权项

1. An integrase-defective lentiviral (IDLV) donor polynucleotide comprising, between the lentivirus LTRs,
a gene of interest and first and second nucleotide sequences that are homologous sequences to a region of interest of endogenous cellular chromatin of a mammalian cell, wherein the homologous sequences are identical to endogenous cellular chromatin sequences of the mammalian cell; and further wherein the donor polynucleotide is selected from the group consisting of: (i) a donor polynucleotide comprising the gene of interest flanked by the first and second homologous sequences and a selectable marker outside the first and second homologous sequences; (ii) a donor polynucleotide comprising the gene of interest, a first selectable marker, and a second selectable marker, wherein the gene or interest and the first selectable marker are flanked by the first and second homologous sequences and further wherein the second selectable marker is outside the first and second homologous sequences; (iii) a donor polynucleotide comprising the gene of interest and a selectable marker, wherein the gene of interest and the selectable marker are outside the first and second homologous sequences; and (iv) a donor polynucleotide comprising the gene of interest and a selectable marker, wherein the gene of interest and the selectable marker are flanked by the first and second homologous sequences.