Patent ID: 11898148
Assignee: CARRYGENES BIOENGINEERING, LLC
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for sequential loading of multiple nucleic acid sequences, each nucleic acid sequence containing at least one gene of interest onto an autonomously replicating nucleic acid sequence having multiple site-specific recombination target sites by removal and reuse of a single recyclable selectable marker gene cassette having an unlimited number of recyclings, the method comprising: in a population of animal or plant cells carrying the autonomously replicating nucleic acid sequence, using a first recombination system, to integrate a first delivery vector comprising a recyclable marker gene cassette and a first gene of interest into a first of the multiple site-specific recombination target sites on, the autonomously replicating nucleic acid sequence;
with a second recombination system and using signal sites alpha, excising the recyclable marker gene cassette from the autonomously replicating nucleic acid sequence, wherein the first gene of interest remains on the autonomously replicating nucleic acid sequence; identifying a first group of cells from the population, wherein the first gene of interest has been loaded onto the autonomously replicating nucleic acid sequence selectable and the recyclable marker gene cassette is no longer present; in the identified first group of cells, using the first recombination system, integrating to integrate a second delivery vector comprising the recyclable marker gene cassette and a second gene of interest into a second of the multiple site-specific recombination target sites on the autonomously replicating nucleic acid sequence; and
with the second recombination system using signal sites beta, excising the recyclable marker gene cassette from the autonomously replicating nucleic acid sequence, wherein the second gene of interest remains on the autonomously replicating nucleic acid sequence.