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

Claim 11:
12. 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 synthetic chromosome 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 synthetic chromosome, 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 recombination target sites on the autonomously replicating synthetic chromosome;
with a second recombination system using signal sites alpha, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the first gene of interest remains on the autonomously replicating synthetic chromosome;
identifying a first group of cells from the population, wherein the first gene of interest has been loaded onto the autonomously replicating synthetic chromosome and the recyclable marker gene cassette is no longer present; in the identified first group of cells, using the first recombination system to integrate a second delivery vector comprising the recyclable marker gene cassette and a second gene of interest into a second of the multiple recombination target sites on the autonomously replicating synthetic chromosome; with the second recombination system using signal sites beta, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the second gene of interest remains on the autonomously replicating synthetic chromosome;
identifying a second group of cells in which the first and the second genes of interest have been loaded onto the autonomously replicating synthetic chromosome and the recyclable marker gene cassette is no longer present;
in the identified second group of cells, using the first recombination system to integrate a third delivery vector comprising the recyclable marker gene cassette and a third gene of interest into a third of the multiple site specific recombination target sites on the autonomously replicating synthetic chromosome; and with the second recombination system using signal sites gamma, excising the recyclable marker gene cassette from the autonomously replicating synthetic chromosome, wherein the third gene of interest remains on the autonomously replicating synthetic chromosome.