|CRISPR/Cas9 Gene Knock-out Cell Lines Construction: Real One-stop Service|
|• Real one-stop service is provided from gene synthesis, design of gRNAs, cell transfection, preparing of single clones to sequencing analysis
of single clone, only needing customers to present target genes information.
• Our CRISPR genome editing technology applies to targets of any genes and mammalian cells.
|• Professional researchers have rich cell culture experience and plentiful CRISPR genome editing experience, including gRNA design, cell
transfection, monoclonal cell culture and passages, solving difficulties in transfection and constructing tumor cell lines.
• Do not add antibiotic to select cells or in cell culture in gene knock-out project. Ensure aseptic operation and sterile environment strictly.
• The gene-editing monoclonal cells and the detailed project reports will be delivered to customers after services are finishes.
|Knock-out cell lines construction||Provided by customers||Contents of delivery||Delivery time|
|Lentivirus-mediated or transient transfection||• Target genes information
• Cell lines
Extra fee will be charged if cell lines are provided by us (general cell lines and 200 tumor cell lines all can be offered)
|• Single cell clones
• gRNA target sequences
• Sequencing reports of target regions
in the transfected cells
It's depending on complexity levels of the target regions and growing states of the cell line at last.
|Cell pre-culture and trying transfection methods||Pre-experiment
• Choose transfection methods of the specific cell: Try all kinds of methods, including liposome-mediated transfection, electroporation, nucleofection, lentivirus-mediated transduction, et al.
• Determine antibiotic concentration: Decrease difficulties in selecting positive cells and checking them.
• Culture monoclonal cells: Determine if monoclonal cells are cultured.
|gRNA design: construct and plasmid preparation||Design gRNA
• Three gRNAs are designed in common exons of different transcriptional products, generally.
• Target positions are as far as possible in the first third of the gene, and targets can destroy important domains of the protein and all the alternatively spliced transcripts.
• Six gRNAs are designed if the effects are not good or the time is pressing.
|Construct vectors or pack lentiviruses
Common Cas9 vectors or lentiviral vectors are selected according to the results of pre-experiments, then vector plasmids and lentiviruses are prepared.
with gRNA, Cas9
Transfect cells with vector plasmids or transduct cells with lentivirus.
|Cell pool analysis: PCR, sequencing for knock-out||Analysize cell pool
Genomic PCR amplicons are sequenced to confirm if genome editing is produced.
|Single clone generation||Select single clones and bank positive cells
Single positive clones are selected according to the sequence results and these cells are banked.
|Single cell clone sequencing and cell banking|
|Final results: single cell clone and full reports||Present final data
Provide clients with the single cell clones and the detailed reports.
|Additional single clones||The other single clones are selected from the same cell bank and presented to clients with analysis of target sequences included in final report.|
|Functional identification of single clones||qPCR and western blot of the target protein.|
1. Target gRNA sequences are designed (free of charge), synthesized and cloned into any vectors according to the target sequences provided by customers.
2. Provide construction service of stable overexpression cell lines and help you to develop stable cell lines expressing foreign genes.
In order to provide quotation efficiently and accurately, please download and complete the inquiry sheet, then fill out the whole inquiry sheet and email it to us, we will reply to you in time.
Download and complete the request form.
The product can only be used for scientific research purposes, NOT be used in any human or clinical experiments, NOT be used to modify any human reproductive system, including the human embryo and reproductive cells.
Knock out the Gln synthysis-related gene in CHO-K1 cells
1. Genomic sequencing results of Gln synthysis-related gene KO cell lines
2. qPCR analysis of Gln synthysis-related gene KO cell lines
3. Growth results of Gln synthysis-related gene KO cell lines
|Genome editing--Knock out target genes
Genome editing refers to an emerging branch of biotechnology that is earlier genetic engineering technologies. By using these biotechnologies researchers are able to target specific DNA sequences and induce a double stranded break, taking advantage of recombination to create synthetic genetic genome of a host.
The RNA-guided DNA endonuclease Cas9 associates with a synthetic single guide RNA (gRNA) and cleaves double-stranded DNA targets complementary to the guide RNA. The double-stranded break occurs 3 bp upstream of the PAM site, allowing targeted sequence modifications via the non-homologous end joining (NHEJ) repair pathway that introduces frame shift insertion and deletion (indel) mutations leading to loss-of-function alleles at the targeted locus.
At present, the CRISPR-Cas9 system has been applied to a variety of model organisms, including human, mice, rats, zebrafish, elegans, plants and bacteria.