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Reporter Genes Knock-In Cell Line Generation

Stable cell lines carrying a reporter gene (a tag or a fluorescent protein) cassette using CRISPR targeted genome editing via the HDR pathway is a powerful tool for monitoring endogenous target gene expression or biological processes without disrupting the target gene in vitro.

Based on years of experience and in-depth investigation, scientists at Creative Biogene can target specific DNA sequences and induce a double stranded break, taking advantage of recombination to create synthetic genetic genome of a host. An expression cassette containing reporter and resistance genes was knocked into the safe-harbor site or any other sites in host cells. Gene insertion at a safe-harbor site allows stable gene expression without any adverse effects on the fitness of the engineered cells.

Applications

  1. Monitoring endogenous target gene expression
  2. Monitoring biological processes such as cell differentiation [1]

Advantages

  1. A wide range of selection of host cell lines, including HEK293 cells, CHO cells, HeLa cells, other tumor cells, mouse ES cells, human ES cells, primary cells etc.
  2. A wide range of selection of reporter genes, including GFP, RFP, His, Flag etc.
  3. Site-specific integration at pre-defined loci, including permissive loci HPRT, Rosa26 and AAVS1 or any other sites.
  4. High integration efficiency with no disruption of endogenous genes.

Reporter Genes Knock-In Cell Line Generation Service Case

1. Knock-in Red Fluorescent Protein (RFP) gene into HEK293 cell line at the Rosa26 safe harbour site.

2. PCR detection of the genomes of transfected cells.

3. Sequencing results of the Rosa26 locus in RFP-positive HEK293 cells
    Sequencing results of the upstream region of Rosa26 site.

Reference

  1. Kimura Y, Oda M, Nakatani T, et al. CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells[J]. Scientific reports, 2015, 5.

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