Gene Editing FAQs

Technical Support

1) What methods do you use to generate genetically engineered cell, mouse and rat models?

We use an embryonic stem cell (ES) based traditional platform to generate mouse models and  CRISPR/Cas9 based Extreme Genome Editing (EGE™) technology to generate cell, mouse and rat models.

 

2) What is the Extreme Genome Editing (EGE™) system?

CRISPR/Cas9 technology,  based on targeted cleavage of the genome followed by homologous recombination, is  widely used for gene editing. However, homologous recombination efficiency of target DNA mediated by conventional  CRISPR/Cas9 technology is low. Through a series of optimizations, Biocytogen has developed an innovative system called Extreme Genome Editing (EGE™).  Compared to CRISPR/Cas9 , EGE™ technology increases  homologous recombination efficiency up to  20 fold and significantly increases the  size of fragments that can be inserted  in the genomes of mice, rats and cell lines.

 

3) How many projects you have completed using EGETM technology?

Biocytogen has used the EGE™ system to deliver more than 2,500 projects worldwide. These models include knockout, conditional knockout, knock-in, point mutation, conditional point mutation and reporter mice. EGE™ ­ is also ideal for gene editing in rats and cell lines (human ES cells, iPS cells and cancer cell lines).

 

4) Which animal strains does Biocytogen use for gene editing projects? Can I request strains not listed here?

Biocytogen  generates  customized animal models using the following strains:

Mice: C57BL/6(J/N), BALB/c , NOD, NOD-SCID, B-NDG, DBA/2

Rats: Sprague-Dawley, Wistar

Please inquire about other strains

 

5) How are F0 (founder) mice validated?

We perform PCR and DNA sequencing to check F0 mice. The whole insert and flanking homology regions are sequenced. F0 animals are then bred with wildtype mice to generate F1 heterozygous mice.

 

6) What are your gene editing service deliverable?

For both EGE™ and ES platform, we deliver one breeding pair of F1 germline heterozygous C57BL/6N mice. Prior to delivery, we perform PCR and DNA sequencing. As an added layer of quality control, we use Southern blots to check for random insertions, and therefore only provide “clean” mice.

 

7) How to eliminate random insertion of the targeting vector?

Some random insertion can be bred out in subsequent generations. Random insertions can be  detected by Southern blot. We have integrated Southern blot in our work flow  as an important quality control step  in  the  generation of knockin and conditional knockout animal models.  Our data shows that the probability of random integration is about 20% for ES cell based gene targeting compared to  35 % chances of random integration in case of CRISPR/Cas9.

 

8) How does Biocytogen minimize off target cutting when using the EGE™ method?

Biocytogen uses bioinformatics approaches to minimize off-target activity by searching the genome for regions of similar sequence identity to the sgRNAs. Therefore, only sgRNAs with high specificity and high activity are chosen.  If off-target effects are of concern for a particular project, we can perform off-target analysis that includes using PCR to amplify the genomic regions around potential off target sites and sequencing them to look for off-target events.

 

9) How is Biocytogen’s animal model service different than other vendors?

Biocytogen provides quality service and excellent project management. From initial project design to delivery of F1 mice, we are here to help you every stop of the way. Our efficient EGETM system is ideal for a variety of mouse genetic models and allows for large-fragment (up to 6 kb) insertions. Our strict quality controls (sequencing/Southern blots) ensure that you receive only the highest quality mice!

 

10) How can I check the status of my project?

When projects are initiated, you will be assigned a project manager, and they will give you monthly updates on the status of your model. Even better, they can be contacted at any time!

 

11) How are payments arranged?

Our payment policy is pretty flexible. You have 2 main options. Biocytogen collects a 30% pre-payment when projects are initiated, and the remainder (70%) when animals are set to be delivered. Alternatively, milestone payments (e.g. targeting vector construction) can also be made. Importantly, there are no hidden fees.

 

12) What happens if my institution cannot accept live mice?

No problem! Biocytogen can also ship cryopreserved sperm

 

13) My institution does not consider Biocytogen as an approved vendor, what are my options?

Biocytogen recently formed a partnership with the Charles River Laboratory (CRL). We are able to ship mouse models to CRL for either breeding or rederivation services.

 

14) After Biocytogen delivers my mouse model, what if I encounter issues establishing my colony?

Here is our backup animal policy:

  1. Live animals will be maintained for 2 weeks after shipment. After 2 weeks, backup mouse strains will be established via frozen sperm and then animals will be sacrificed. The sperm can be stored for 6 months for free and will cost $800/3 years/line to store thereafter.
  2. If within 45 days the customer reports unhealthy animals that resulted in the shipment, we will rederive the animals and ship for free. Otherwise, the rederivation and shipping fee is $4,500/line.

 

15) What are the advantages of gene targeting by ES cell  versus CRISPR/Cas9?

Targeting via ES cells allows for complex targeting and for making insertions greater than 6 kb in the mouse genome.

 

16) What is the genetic background of mice generated from ES cells?

Biocytogen’s ES cells are 100% pure C57BL/6 background. This can save up to 2 years back cross time!

 

17) How are transfected ES cells screened? How can I ensure the correct target gene insertion and karyotype changes in long-term in vitro culture of ES cells?

During targeting vector construction, a positive selection marker (NEO resistance gene) and a DTA negative selection marker  are introduced; at the same time, a restriction enzyme site is added  to a specific position in the targeting vector. After electroporation of the targeting vector into the ES cell, G418 selection is used to screen for positive cells. . We also perform Southern blot to ensure that there are no random insertions. In addition, we monitor karyotype changes in ES cell subjected to long-term in vitro culture by performing karyotype analysis. Using chrsomosome banding technology, the chromosomes are analyzed, compared, sequenced and numbered according to chromosome length, centromere position and arm ratio, ensuring that subsequent micro-injection is performed only after high-quality ES cells is obtained.

 

18) Which factors influence gene targeting in ES cells and what is the success rate for projects?

The most important factor in gene targeting is the strain of the mouse embryonic stem cell; it is easier to target ES cells in some strains compared to others. The number reported in literature is 0.5-3%, but it is easier to target at some loci, such as the Rosa26 locus, with significantly higher efficiency. Gene targeting efficiency is relatively high, if recombination is performed with embryonic stem cells from C57BL/6.

 

19) Can you gene edit cell lines?

Yes, we routinely perform gene knockout, knock-in point mutation, and large fragment knock-ins using ATCC cell lines, mouse and human ES cells, and human iPS cells.

 

20) Can you gene edit cell lines provided by customers?

YES – but it will take longer than the typical turnaround. If we haven’t used the cell line in house for  gene editing, you can ship your cells to us!, We need to perform preliminary testing first which includes mycoplasma detection, karyotyping, antibiotic titration, transfection efficiency optimization and colony formation assay. This process takes around 4 weeks.

 

21) What are the cell line project deliverables? What steps are performed to validate the final single cell clones?

For a knockout cell project, we deliver two homozygous KO cell clones  and for knock-in cell projects, we deliver two  heterozygous KI cell clones. All clones are validated by two rounds of PCR and DNA sequencing. We also provide a negative control cell line (electroporation of a control CRISPR/Cas9 plasmid pCL-NC, encoding the Cas9 nuclease and a non-specific guide RNA to wildtype cell line, without any drug selection).

Services

1) What services do you provide?

We perform gene editing services using mice, rats and cell lines. We also use a Tol2 transgenic system to make transgenic mice and rats.

2) What is the general timeline of EGE™ system vs. ES cell-based homology recombination strategy (ESC/HR)?

EGE™ system: 6-8 months for F1 germline transmission mice

ESC strategy: 9-11 months for F1 germline transmission mice

3) What information is required from the customer at the start of a project?

Customers only need to provide the name of gene of interest and other pertinent information (e.g desired mutation, tag). We will make professional evaluations and generate design(s). We also perform a sequence analysis for the gene prior to providing a preliminary design. Gene editing specialists will correspond with you  via email, phone chat, or web-based conference. After a consensus is reached, your gene editing specialist will issue a service agreement, and projects will be initiated.. Biocytogen’s production team will then generate and deliver your animal or cell model.

4) How are communications handled, and how can I track the progress of my project?

Every project will be assigned to a senior scientist & project manager who will report to you monthly about the progress report with all the original data in it. Technical support team is available all the time for any questions you might have.

5) How do you ensure that the generation of mouse models and their husbandry meet acceptable standards?

We have a high standard validation system. Germline transmission F1 heterozygous mice which will be delivered to you will be fully validated by two rounds of PCR, Southern analysis, and DNA sequencing in order to screen out the possibility of random insertion and ensure the targeted locus has 100% correct DNA sequence.

Biocytogen’s animal facility is fully and continuously accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC #001662) and assured by US Public Health Service (USPHS #A5994-01).

Sentinel program is applied. The health-monitoring (HM) program on sentinel samples is performed quarterly according to Charles River comprehensive panel.

6) What if the project failed?

Based on the past 3000+ mouse models we have generated, our success rate is about 98.6%. If in rare cases the project fails or it appears that it may be delayed, your project manager will discuss with you ahead of time to find an alternative way to generate your model. For example, if an EGETM based project fails, it can be concerted to an ES based project. If it still doesn’t work, we will provide you a 100% refund. This 100% guarantee policy is strictly stated in the service agreement.

7) What’s your backup plan?

Live animal can be maintained for 2 weeks after shipment. After 2 weeks, live animal will be sacrificed and we will keep the backup mouse strain by frozen sperms for 6 months for free. We charge $800/3 years/line for storage of the mouse sperm thereafter.

Within 45 days after shipping, we can arrange another shipment for free if the customers report the bad health status due to the shipment.

The rederivation and shipping fee is $4500/line if the line is lost due to customers’ reason.

8) Do you provide a genotyping strategy?

Yes, we will provide genotyping results of F1 heterozygous mice and a genotyping report which includes a protocol.

9) Do I have to use the same genotyping reagents that are listed in your protocols?

No, the protocols can work with other reagents. You may need to optimize the conditions for the assay to work in your lab.

10) Do you have construct or sequence information used to develop the mouse so I can develop new primers?

Yes, we can provide the construct or sequence information to you. We can also help you to design other sets of primers.

Shipment

1) Where can Biocytogen ship the live animals to?

Biocytogen ships custom animal models to many countries including the United States, Europe, Australia, and Asia. Some countries have specific regulations regarding the shipment of biological materials, but we will work with you to deal with these issues.

2) Who has access to Biocytogen animal facility and barrier?

Veterinarian and animal facility personnel.

3) What strain and age are the sentinels?

C57BL/6N, 1-4 months (4-5 week set, 1-4 months after sent out for test)

4) Which lab performs the health surveillance (serology, bacteriology and parasitology)?

Charles River Laboratories (CRL)

5) Will the animal you deliver be SPF grade?

Yes, it’s the same standard as Charles River.

6) How long have the animal facilities at Biocytogen maintained their health status?

Beijing Biocytogen Co. Ltd: since May 2015

Biocytogen Jiangsu Co. Ltd: since December 2015

Biocytogen Boston Corp: since January 2018

Payments and IP

1) What’s the payment structure?

For customers in academia, Biocytogen collects a 30% prepayment when projects are initiated. The remaining balance is due when final products are ready to be shipped out. Alternatively, milestone payments (e.g. targeting vector construction) can also be made.

For industry customers, we collect a 50%  prepayment. The remaining balance is due when final products are ready to be shipped out.

2) What payment methods can customers use?

US clients must remit payment by check and wire transfer. Non-US clients must remit payment by wire transfer.

Remit Address:

Pay to: Biocytogen Boston Corp.

Mail check to: Biocytogen Boston Corp. Attn: Account Receivable. 50C Audubon Road, Wakefield, MA 01923, USA

3) Who owns the intellectual property (IP) of the animal model?

Customers own the intellectual property of the customized animal model.

Back to top