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Services

 Services; iPS, CRISPR, Virus, Cloning


 

iPS

Fibroblast derivation:

include deriving, expansion, and freezing of fibroblasts from freshly collected skin biopsy, mycoplasma tested, at least 500k/vial x 3 vials.

mRNA-based reprogramming from human fibroblast cells:

  • Reprogramming is done with feeder free system.
  • Picking at least 12 colonies
  • Expansion of top three to four colonies
  • Deliver 2 clonally expanded mycoplasma free iPSC lines  at p5-p7(at least 500k/vial x 3vials) with basic characterization, including cell authentication, immunostaining (Oct3/4, Sox2,Nanog,Tra160, Tra181).

Sendai based reprogramming from human fibroblast cells/blood

  • Reprogramming is done with feeder free system
  • Picking at least 12 colonies
  • Expansion of top three to four colonies
  • Deliver 2 clonally expanded mycoplasma free iPSC lines at around p7 (at least 500k/vial x 3vials) with basic characterization, including cell authentication, immunostaining (Oct3/4, Sox2,Nanog,Tra160, Tra181)
  • If needed, we can run test for presence of sendai virus, we charge 2000kr/clone/test every 3-5 passage after passage 10

Provide human ES cell lines

We have banked numerous Harvard and WiCell generated human embryonic stem cell lines and have agreements in place to distribute these lines to users via an accelerated MTA approval contract at significantly reduced cost to local users.

Access to control iPS lines

The iPS lines generated in the core may be obtainable to other users depending on the ethical approval agreements under which they were established. We provide a list of iPS lines produced at the facility and act as the intermediate for users to be able to establish contact with those who have the lines. We also help provide advice on how to write the ethical agreements to facilitate a broader use of novel iPS lines in research collaborations.

User access to Cell Culture Clean Rooms

The facility is complete with advanced and state-of-the-art pluripotent stem cell tissue culture equipment, reagents, cell storage facilities all maintained by the core facility staff. Users of the facility must undergo a 1 hour introduction training prior to working in the facility. LAF hoods are booked via online calendar. Users may use the facility in a limited and availability dependent manner. The work in the core is limited to ES and iPS cell culture, maintenance, and expansion; not differentiation of the pluripotent stem cell lines. 


CRISPR

  • Experimental design
  • gRNA design (CRISPR-cut, CRISPRi, CRISPRa)
  • CRISPR edits in iPSCs (new)
  • HDR design and strategies for tagging and correction
  • Cloning protocols for gRNA insertion
  • Cloning & virus production services (see below)
  • Initial validation of CRISPRi and CRISPRa in HEK or iPSC (new)
  • Cut and edit validations strategies
    • Pipeline for NGS amplicon sequencing
    • Referral to TigerQ  (complete service)

Virus production services

Full service production of lentivirus, oncoretroviral and AAV.

Our trained staff will take the vector plasmid you provide us and within 4 weeks you will receive a high titer vector preparation. We have the technical experience as well as state-of-the-art infrastructure to be able to provide users with highly reproducible, high-titer vector preparations using ultra-centrifugation.

Useful Information:

  • Typical concentration and volume achieved for viral preps of GFP containing lentiviral vector is 100uL 10E7-10E8 TU/mL from a single 15 cm dish. 
  • For 10 plates of the same vector we obtain 500ul-1000ul 10E7-10E8 TU/mL.
  • The quality of the DNA matters, please ensure that you have performed restriction digest and/or sequencing and have an OD ratio of 1.7-2.0.

 


Cloning

  • Construct design and cloning strategies consultation
  • Construct building / full cloning service
  • Contruct testing and validation e.g. restriction digestion, sequencing
  • Maxiprep Service (or other DNA scale-up)
  • Related molecular biology services (tell us about your molecular biology needs) 

 

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