US Patent No. 10,655,134

PRODUCTION OF RECOMBINANT EXPRESSION VECTORS


Patent No. 10,655,134
Issue Date May 19, 2020
Title Production Of Recombinant Expression Vectors
Inventorship Ralf Amann, Tuebingen (DE)
Hans-Joachim Rziha, Tuebingen (DE)
Assignee EBERHARD KARLS UNIVERSITAET TUEBINGEN MEDIZINISCHE FAKULTAETGESCHWISTER-SCH...

Claim of US Patent No. 10,655,134

1. A method for the production of recombinant expression vectors, comprising the following steps:(1) providing a cell population containing a starting vector, said cell population expresses a first gene product encoded by the starting vector, whereas in the starting vector the coding nucleotide sequence for the first gene product is upstreamly and downstreamly flanked by first recombination nucleotide sequences;
(2) transfecting the cell population obtained from step (1) with a transfer vector encoding a second gene product, whereas in the transfer vector the coding nucleotide sequence for the second gene product is upstreamly and downstreamly flanked by second recombination nucleotide sequences which are homologous to said first recombination nucleotide sequences;
(3) incubating the cell population obtained from step (2) under conditions which allow an exchange of the coding nucleotide sequence for said first gene product against the coding nucleotide sequence for said second gene product by homologous recombination of said first and second recombination nucleotide sequences and the formation of a recombinant expression vector;
(4) incubating the cell population obtained from step (3) under conditions which allow the expression of said second gene product;
(5) contacting the cell population obtained from step (4) with a first binding molecule that binds to said first gene product under conditions which allow the formation of complexes of said first binding molecule and said first gene product;
(6) separating said complexes of first binding molecule and first gene product from the cell population;
(7) isolating the recombinant expression vectors from the cell population;
(8) incubating the recombinant expression vectors with non-transfected cells under conditions which allow a transfection of the non-transfected cells with said recombinant expression vectors to obtain a further transfected cell population;
(9) contacting said further transfected cell population with a second binding molecule which binds to the second gene product under conditions which allow the formation a complexes of the second binding molecule and the second gene product;
(10) separating said complexes of the second binding molecule and the second gene product; and
(11) repeating the steps (8) to (10) at least one additional time.