Generating stable cell lines with quantifiable protein production using CRISPR/Cas9-mediated knock-in
| Autor: | Chiu-An Lo, Alexander W. Greben, Brian Edwin Chen |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 62, Iss 4, Pp 165-174 (2017) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/000114534 |
| Popis: | Cell lines expressing foreign genes have been widely used to produce a variety of recombinant proteins. However, generating recombinant protein–expressing cell lines is usually a lengthy process and the resulting protein expression levels are often inconsistent. Here, we describe an efficient method for making stable cell lines expressing any recombinant protein of interest in a controllable and quantifiable manner. We integrate transgenes into specific genomic loci using CRISPR/Cas9 such that transgene expression is driven by endogenous promoters to ensure consistent and predictable expression of the recombinant protein. Expression levels can be predetermined by selecting promoters from genes with the desired level of expression. To quantify recombinant protein expression, a protein quantitation reporter (PQR) is incorporated between the endogenous and foreign genes. The PQR allows equimolar production of the endogenous protein, the recombinant protein, and a fluorescent reporter. As a result, expression levels of both the endogenous and recombinant proteins can be continuously monitored using fluorescence. |
| Databáze: | Directory of Open Access Journals |
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