FAQs
Better gene editing, better cell line development, better science.
Thanks to FAST-HDR + CRISPR.
What products and services does ExpressCells offer?
We offer three product lines:
- Fully customized cell lines in which you specify as many as three proteins of interest (POIs) for tagging
- Catalog cell lines in which from one to three POIs are already tagged
- ”Backbone” cell lines where one or two cellular or subcellular structures are pre-tagged and you specify a second or third POI for custom tagging.
HEK293T cells in which Histone 3.3 has been tagged with mRuby3, ATP5B with BFP2, and β-tubulin with mClover3.
Watch ExpressCells most recent seminar on gene editing – "Creating Better Gene-Edited Cell Lines with the FAST-HDR System"
What you’ll learn:
- Understand the current challenges in creating custom gene-edited cell lines
- Know the technology underlying the FAST-HDR gene-editing system, including its use with CRISPR
- Be able to describe the advantages of the FAST-HDR system
- Learn about several case studies using gene-edited cell lines
What products and services does ExpressCells offer?
We offer three product lines:
- Fully customized cell lines in which you specify as many as three proteins of interest (POIs) for tagging
- Catalog cell lines in which from one to three POIs are already tagged
- ”Backbone” cell lines where one or two cellular or subcellular structures are pre-tagged and you specify a second or third POI for custom tagging.
How do you prevent the labeling tag from interfering with the function of the targeted protein?
In the FAST-HDR System, we included a flexible linker sequence that separates the targeted protein from the labeling tag. This approach allows the proteins to be connected and to have spacing to facilitate getting into their correct 3D structure. This approach has worked on labeling essential proteins. However, this approach only partially guarantees that there will be no interference, and sometimes it is required to consider tagging the target protein at the N-term instead of the C-term.
How would you handle a protein with very low expression to accomplish genetic tagging?
We can use a couple of strategies to generate cell lines with tagged endogenous proteins with low expression. Suppose the expression of the protein can be induced with a small molecule or cytokine. In that case, we can use those molecules to increase expression and facilitate selection during the antibiotic selection step. Alternatively, we can use lower doses of the resistant antibiotics to find a dose that will allow the selection of modified cells with low expression levels of antibiotic-resistant proteins.
With what cell lines do you work?
We work with a number of cell lines. Currently, our catalog and backbone offerings are based on HEK293T, U-2 OS, and HeLa cell lines. Examples of other cell lines that we have used include Expi293, HEL92.1.7, OCI-AML3, SNU-899, CHO, and ID8. We have also worked with porcine and bovine cell lines. Please contact us to discuss your cell line options.
How long does it take to deliver a custom cell line with a single reporter knock-in? With 2-reporter knock-ins? With 3-reporter knock-ins?
We can deliver a custom cell line with a single reporter knock-in in as few as 30 days, two reporter knock-ins in as few as 60, and three reporter knock-ins in as few as 90.
Can I order a cell line with up to two organelles pre-labeled and with a single custom knock-in?
Absolutely. In our semi-custom cell line service, we stock backbone cell lines with the following combinations of pre-tagged organelles: nucleus and cell membrane; nucleus and cytoskeleton; nucleus and endoplasmic reticulum; and nucleus and mitochondria. You specify the third POI.
What are the characteristics of ExpressCells’ Cas9/sgRNA plasmid?
We use a plasmid containing a high-fidelity Cas9 with high, on-target¹ specificity/low, off-target activity and an optimized sgRNA that must fully interact with the target sequence before cleavage by Cas9. The plasmid, modified to facilitate cloning of sgRNA oligos, is produced in a single-step reaction with 100% efficiency.
¹ Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351:84-8.
What is the process for creating a custom cell line?
First, we analyze the sequence of the target gene or genes to determine whether it is biologically possible to effect the cut and the insertion where desired and whether it will be possible to synthesize appropriate recombination arms.
Do you design multiple sgRNAs for a single knock-in?
Because we are focused on knocking in specific genes via homologous recombination, we generally don’t have great flexibility in terms of the sequence of the sgRNA. However, if there is more than one CRISPR cutting site in a gene of interest, multiple homology arms and different donor vectors might be created.
How does the FAST-HDR plasmid vector system reduce false positives in the knock-in pool following transfection?
Expression of the antibiotic resistance gene cannot occur without integration. Thus, cells that survive positive selection have been successfully modified.
How do you address low-expression genes?
For HEK293T and HeLa, expression levels for most proteins are known. In certain cases, inducers can be used during clonal selection.
What is the workflow you use to validate targeted insertion of a tag or tags?
We use PCR followed by Sanger sequencing.
Is Western blotting part of your workflow?
Western blotting is an optional add-on to the validation workflow.
I have a proprietary cell line. Can you use it for custom knock-ins?
That depends both on the cell line and the POI. Please contact us to discuss.
What type of cell lines have you been able to modify with the FAST-HDR System?
We have done genetic modifications in multiple cancer cell lines that are adherent or grow in suspension. The preferred cell lines have doubling times <30 hours and are a good host to incorporate plasmid DNA with either transfection or electroporation. In addition to working with human cell lines, we have also worked with mouse, rat, and hamster cell lines.
What is the process for ordering a custom knock-in?
Please complete the Contact form below and we will be in touch.
What are the deliverables?
- 1 clone of KI cells (2 vials: ≈ 5 x 105cells each)
- 1 vial WT cells (≈ 5 x 105cells)
- Milestone reports sent periodically
- Project report
- Milestone-based project report
- Sequence of guide used
- Primer sequences for PCR and sequencing
- Certificate of Analysis of cells
- Mycoplasma (Pass/Fail)
- Genotype
What tags are available?
Luciferase, mClover3, mRuby3, and mTagBFP2 are standard. Other tags are available by special order.
Do you perform C-terminal or N-terminal tagging?
Where practicable, we prefer C-terminal tagging since that is less likely to affect endogenous expression of the POI.
In the case of C-terminal tagging, what is the chance of the tag and resistance program being expressed via off-target integration?
Minimal, in that both need the endogenous promoter and the tag needs to be in-frame to be functional.
In the case of C-terminal tagging, is there a possibility of non-homologous end-joining with indels in one allele of the gene of interest or elsewhere in the genome?
It is indeed possible but unlikely to be biologically important since cuts are generally made at the very end of the protein sequence, and the protein will be fully formed until that point.
Can you generate homozygous knock-in cell lines?
Yes, in those cases in which function of an essential protein would not be adversely affected by the fluorescent tag.
Does this affect your timelines?
Only modestly, since transfection with two donor vectors bearing different resistance genes is done simultaneously.
Is Southern blotting used to rule-out off-target integration of a reporter in the case of custom knock-ins?
We consider the likelihood of aberrant expression of a reporter gene in the context of off-target insertion of that gene to be very low. Thus, Southern blotting is not typically part of our validation work-flow. It can, however, be added to that workflow as an option, adding several days to the validation process.
What is the timeline to deliver catalog cell lines?
Please complete the Contact form below and we will be in touch.
What is your payment policy?
For custom and semi-custom cell lines, we require a non-refundable 20% deposit at the time of ordering, payable via credit card or ACH. The balance is invoiced upon delivery of the cell line to the customer. Catalog cell lines are invoiced upon ordering and delivery.
Better knock-ins. Better cell lines. Better science.
