Single Gene Knock Out Cell Line Generation

Service ContentAdvantagesProduct DataCase Study

Service Content

CRISPR/Cas9 is a revolutionary technology in the field of gene engineering. It has expedited the course of biopharmaceutical research and drug discovery, by providing scientists with a versatile tool to knockout any gene, in any cell and without introducing foreign DNA. The benefits of CRISPR/Cas9 over previous forms of gene editing, such as TALENs and zinc finger nuclease (ZFN), are that it is much simpler to implement and has higher efficiency at performing bi-allelic gene modifications.

Creative Biogene is experienced to provide a full CRISPR/Cas9-based single gene knockout service using any mammalian cell line and targeting any gene. Our scientists are experts at performing gene knockout with CRISPR/Cas9, from designing gRNA constructs to transfection and single clone generation of a wide range of cells, including difficult-to-transfect and tumor cell lines.

Our services include:

Host cell characterization
- Optimize transfection methods of the host cell: Try all kinds of methods, including liposome-mediated transfection, electroporation, lentivirus-mediated transduction, et al.
- Test the minimal absolute lethal dose of antibiotic concentration.
- Test the clonability of the host cell: Determine if monoclonal cells are cultured.
gRNA design and KO vector construction
- gRNAs are designed in common exons of different transcriptional products, generally.
- Targets can destroy important domains of the protein and all the alternatively spliced transcripts.
Transfection
- Transfect cells with the optimal transfection methods.
Single cell screening
- Select cell by MACS, FACS or antibiotic selection and screen gene-edited cell clones.
- Expand the engineered cells.
Validation
- Evaluate the target stable cell clones via one or several assays such as Western Blot, ELISA, real-time PCR, or reporter assays etc.

Advantages

One-stop service is provided from design of gRNAs, cell transfection, preparing of single clones to sequencing analysis, only needing customers to provide target genes information and the cell line of interest.
Our CRISPR gene knockout technology applies to targets of any genes and mammalian cells.
Professional researchers have rich cell culture experience and plentiful CRISPR genome editing experience.
The detailed project reports will be delivered to customers after services are finishes.
Cell lines can be made from nearly any cell type, including cells that are difficult to transfect and grow incl. primary cells.

Product Data

Product Data 1: CD47 Knockout Cell Line-HEK293T

Figure 1. (A) CD47 Knockout Cell Line-HEK293T. (B) Cleavage site sequencing analysis. (C) Flow cytometry analysis.

Product Data 2: B2M Knockout Cell Line-K562

Figure 2. (A) B2M Knockout Cell Line-K562. (B) Cleavage site sequencing analysis.

Product Data 3: Human EGFR Knockout Cell Line-A549

Figure 3. (A) Human EGFR Knockout Cell Line-A549. (B) Cleavage site sequencing analysis.

Case Study

Case Study 1

Figure 4. Deletion of TFAP2A gene significantly reduces TGF-β1-induced fibroblast differentiation. (A) CRISPR/Cas9-based gene editing in NIH/3T3 fibroblasts deleted the TFAP2A expression, as validated by pooled Real-time PCR and immunoblotting. (B-E) Gene expression of α-smooth muscle actin (α-SMA), collagen (COL) 1A1 (COL1A1), COL2A1, and COL3A1 were quantitatively analysed by real-time PCR in wild type and TFAP2A-KO fibroblasts. (Ross G R, et al., 2019)

The TFAP2A knockout cell line with NIH/3T3 fibroblasts (TFAP2A‐KO) was established using CRISPR/CAS9 technology through Creative Biogene, Shirley, NY.

* For research use only. Not intended for any clinical use.

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