Cell surface tag exchange (CSTE) system for tracing and manipulation of cells during recombinase mediated cassette exchange integration of nucleic acid sequences to engineered receiver cells

A method for generating derivative engineered cells expressing a TEDV-encoded gene from a TERS, said method comprising,

• generating a TEDV encoding a first CST exon and a gene and containing no promoter sequences;
• delivering said TEDV to an engineered cell line containing a paired TERS encoding a second CST exon and also encoding a reporter gene in an antisense orientation, along with a recombinase enzyme, wherein the recombinase enzyme mediates cassette exchange between the matching RMCE elements encoded in the TEDV and the TERS;
• contacting at least one cell of the engineered cell line with two or more affinity reagents specific for both the TEDV-encoded CST exon and the TERS-encoded CST exon;
• selecting at least one derivative engineered cell on the basis of diminished expression of the reporter gene and TERS-encoded CST exon, and increased expression of the TEDV-encoded CST exon.

A method for generating multiple derivative engineered cells expressing a range of TEDV-encoded gene, from a pool of TEDVs, said method comprising,

• generating a library of two or more TEDVs, each encoding a unique gene sequence, each with a unique TEDV-encoded CST exon, and each containing no promoter sequences;
• delivering said library of TEDVs as a pool to an engineered cell line containing a paired TERS encoding a unique CST exon and also encoding a reporter gene in an antisense orientation, along with a recombinase enzyme, wherein the recombinase enzyme mediates cassette exchange between matching RMCE elements encoded in the TEDVs and TERS;
• contacting cells with three or more affinity reagents specific for both the multiple TEDV-encoded CST exons and the TERS-encoded CST exon;
• selecting at least one derivative engineered cell on the basis of diminished expression of the reporter gene and TERS-encoded CST exon, and increased expression of each of the unique TEDV-encoded CST exons.