Patent ID: 9365864
Filing Date: 2016-06-14
CPC Classification: C12N

Claim Text:
1. A method for transforming by homologous recombination at least one cell in vitro, comprising: (a) stably transforming at least one cell by inserting construct (i), which is encoded by a nucleic acid molecule, which comprises: wherein A1 is a first promoter, A2 is a first homologous portion, A3 is a meganuclease cleavage site, A4 is a first marker gene, A5 consists of the neomycin resistance gene of SEQ ID NO:7, and wherein construct (i) is configured to be stably integrated into the genome of at least one target cell, into the genome of said at least one cell; (b) cloning a sequence coding for a gene of interest into position B3 of construct (ii), which is encoded by a nucleic acid molecule, which comprises at least the following components: wherein A2′ comprises a portion of said first homologous portion A2, B1 is a second marker gene different from said first marker gene, B2 is a second promoter, B3 is a multiple cloning site, B4 is a third promoter, A5′ consists of the inactive neomycin resistance gene of SEQ ID NO:13; (c) co-transfecting said cell of (a) with said construct (ii) of (b) and construct (iii), (iv), or (v), which are encoded by nucleic acid molecules, which comprise components: an isolated or recombinant protein which comprises component: wherein C1 is a fourth promoter, C2 is the open reading frame (ORF) of a meganuclease, C3 is a messenger RNA (mRNA) encoding said meganuclease, and C4 is an isolated or recombinant protein of said meganuclease, wherein a meganuclease from construct (iii), (iv), or (v) recognizes and cleaves A3, and construct (iii), (iv), or (v) are configured to be co-transfected with construct (ii) into said at least one target cell; (d) following homologous recombination between said construct (ii) and said stably inserted construct (i), selecting at least one cell from (c) based upon: the absence of a first marker gene encoded by component A4 of said construct (i) and the activity of a second marker gene encoded by component B1 and neomycin resistance activity.