The Gene Editing Institute specializes in complex genetic engineering with the technical expertise, facilities and technology to create genetically modified cell lines tailored to our client’s precise requirements.
We design our own gene editing tools in-house, using TALENs, CRISPR-Cas9 and synthetic ssODN systems, to permanently disrupt or knockout genes, add or knock in DNA fragments and/or create point mutations in genomic DNA. In many cases, we also produce gene targeting and transgenic vectors in house, which can be tailored to designs provided by individual laboratories.
We have worked successfully with organizations such as The Wistar Institute of Philadelphia, the Mayo Clinic, Dartmouth University, The University of Vermont, the University of North Dakota and Thomas Jefferson University, among many others.
How to begin the process
- We request that each client provide an NCBI Gene ID# for the gene that is targeted for alteration.
- Clients may supply us with their cell line of choice along with their preferred culture conditions and transfection methods.
- When appropriate, our team uses Sanger sequencing to characterize the gene target in the client’s cell line, and bioinformatics to identify gene copy number and SNPs.
- We also analyze the target for various gene isoforms that generate altered or truncated proteins to ensure all possible gene copies are effectively targeted.
Success depends upon the ability to accurately identify the gene copy number, genetic variance and possible isoforms, as these characterizations may impact the efficiency and difficulty of carrying out successful gene editing reactions. Failure to identify and account for isoforms, during CRISPR design for example, can lead to functional protein variants even after frame-shift mutations are created.
That is why, prior to the start of workflow, we provide data on gene copy number, genetic variance and isoforms to our clients.
Cell line modification
- Cell line modification begins by transfecting the cells with an indicator gene or fluorescent tag. This allows us to employ FACS (fluorescence-activated cell sorting) to observe and enrich the population of tagged cells.
- Next step is to isolate a sampling of that cell population to determine the extent of gene editing activity. This is accomplished by direct Sanger sequencing of the target region, followed by analysis using Tracking of Indels by DEcomposition (TIDE) software.
- We provide clients promptly with the results of these analyses to calculate an estimate for the necessary number of clones to be screened.
It is important to note that the expansion of isolated modified cell lines in sufficient quantity for shipment is completely dependent on the speed at which the targeted cell lines divide.
Allowing for cell growth rates, our standard single cell cloning protocol will generate transfected clonal cell isolates in about six weeks. During this process, our team communicates closely with clients and provides weekly updates on progress toward final clonal isolation and verification.
- If cell morphology changes are observed after genomic modification, we provide images documenting these changes as part of the update process.
- Once purified DNA samples from isolated clones have been analyzed, we provide a vial of the modified cell line to the client directly.
- For each final clone, we prepare a TIDE allelic analysis noting the exact type of genetic modification that has occurred within the clonal isolate at each gene loci.
Our methodical approach typically generates a series of various heterozygote and homozygous mutated cell lines. In some cases, these have proven valuable for observing gene dosage effects, avoiding lethal effects, etc. Any and all analyzed clones may be provided to the customer as part of the order.
Costs
Unlike other commercial operations, The Gene Editing Institute does not charge a higher price point for using the client’s preferred cell lines. We do all custom work for all cell lines at the same price.
Costs are determined after our initial analysis of the target site and the host cell line. Individual cell line characteristics may lead to some variation in costs for cell line modification.
In general,
- Generation of a knockout cell line ranges between $5,000 and $10,000.
- Multiplexing is available, where up to 5 genes are knocked out simultaneously or in various combinations within the same cell line. Costs are negotiated based on the number of candidate genes and the particular cell line.
- Generation of a knock in or point mutation cell line is approximately $10,000.
- A discount (up to 25%) may be applied for individual orders of 10 or more cell lines.