公开(公告)号：US11802281B2

公开(公告)日：2023-10-31

申请号：US16090829

申请日：2017-03-17

Applicant: ETH ZURICH

Inventor： Sai Reddy ,  William Kelton ,  Cristina Parola ,  Derek Mason ,  Mark Pogson

IPC: C40B20/04 ,  C12N15/10 ,  C12N5/0781 ,  C12N9/22 ,  C12N15/113 ,  C40B10/00 ,  C40B40/08

CPC classification number: C12N15/1037 ,  C12N5/0635 ,  C12N9/22 ,  C12N15/102 ,  C12N15/1058 ,  C12N15/1082 ,  C12N15/1086 ,  C12N15/113 ,  C40B10/00 ,  C40B20/04 ,  C40B40/08 ,  C12N15/1034 ,  C12N2310/20 ,  C12N2510/00 ,  C12N2510/02 ,  C12N2800/80 ,  C12Q2521/539 ,  C12N15/102 ,  C12Q2521/539

Abstract: According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.