Motomi OSATO

Transgenic and Gene Targeting Facility

The Transgenic & Gene targeting facility is located in Clinical Research Centre (CRC) at MD11 in NUS. The facility is equipped with Leica AM6000 micromanipulator, Eppendorf FemtoJet micro-injector, micro-pipette fabrication devices and tissue culture system.

Fluorescence Activated Cell Sorting (FACS) Facility

The Facility currently operates 2 Becton-Dickinson FACSAria II for cell sorting and analysis. These machines have 3 or 5 air-cooled lasers and are capable of performing 9 – 12 colour flow cytometry analyses, 2- and 4-way bulk sorting to tubes, and single-cell sorting to well plates (ranging from 6 to 96 wells) or microscope slides.

Transgenic and Gene Targeting Facility

Background

The Transgenic and Gene Targeting Facility (TGTF) is located at Clinical Research Centre (CRC), MD11, in NUS. The facility is equipped with Leica AM6000 micromanipulator, Eppendorf FemtoJet micro-injector, micro-pipette fabrication devices and tissue culture system. The facility provides services and collaborative supports in generating genetically-engineered mouse (GEM) models and cryopreserved sperms and embryos, for basic and translational biomedical research. The following services are currently offered by the facility:

Services

Generation of genetically-engineered mouse (GEM) by CRISPR/Cas 9 technology

TGTF has successfully implemented CRISPR/Cas9 technology to generate gene targeted animal models for mice and rats by pronuclear microinjection and electroporation (upcoming). The advantages of CRISPR/Cas9 genome editing for the creation of unique mouse models include time-savings, potential reduction in animal usage, and overall cost effectiveness. The full services include molecular design, guide RNA validation, pronuclear microinjection or electroporation, embryo transfer to surrogate mothers, and care of newborn mice till weaning. Customers should be responsible for genotyping to confirm transgenic founders. The turnaround time is around 3-4 months. This service is now available for C57BL/6 strain which was previously very hard to be used for the gene targeting in mice.

Generation of transgenic mice by traditional pronuclear DNA microinjection

This service is also available to meet some customers’ special needs for classical transgenesis by random insertion of a transgene. We guarantee 3 founder transgenic mice for a neutral construct, or injection of 200 zygotes of FVB/N or C57BL/6 strains. Customers should be responsible for genotyping to confirm transgenic founders.

Gene targeting in mouse embryonic stem (ES) cells

Because of their plasticity, embryonic stem (ES) cells are traditionally employed for gene targeting in mice. Mouse ES cells are electroporated with a targeting vector for DNA integration. After colony screening, positive clones are expanded and then injected into a normal mouse blastocyst. The blastocysts colonized with the ES cells are transferred into surrogate mothers and developed into chimeric mice. These mice are then bred with mice to produce animals that have germline transmission of the targeted gene. The turnaround time is around 6-9 months. ES cells to be used in this service include those derived from 129 and C57BL/6 strains. Targeted ES cells from public bioresources or other researchers are also be used in this service.

Sperm and embryo cryopreservation

Any GEM may potentially encounter multiple problems, such as infertility, genetic drift, and loss or acquisition of specific phenotypes, particularly when maintained over the years. To overcome these problems, Assisted Reproductive Techniques (ART) and Cryogenic Techniques (CT) have been employed in the facility.

Benefits of cryopreservation:

  • Prevention of genetic drifts or spontaneous phenotype changes
  • Low cost as compared to live mouse maintenance
  • Allowing for a strain exportation without quarantine

Rederivation

The above-mentioned cryopreserved embryos and sperms can be used for the re-establishment of a particular mouse strain. This procedure is a rederivation by which the embryos are transferred into the reproductive tract of a pseudopregnant mouse in order to allow implantation, gestation, and birth.

Benefits of rederivation:

  • Removing pathogens from your contaminated mouse lines
  • Retrieving your mouse line from cryopreserved sperm or embryos
  • Retrieving your mouse line from old sterile male mice within a period as short as 6 weeks.
  • A convenient way to import mice from a collaborator.

Notes

1. Please contact us (Edward Shi: csisj@nus.edu.sg) for further information and price list.

Fluorescence Activated Cell Sorting (FACS) Facility

Background

The Facility currently operates 2 Becton-Dickinson FACSAria II for cell sorting and analysis. These machines have 3 or 5 air-cooled lasers and are capable of performing 9 – 12 colour flow cytometry analyses, 2- and 4-way bulk sorting to tubes, and single-cell sorting to well plates (ranging from 6 to 96 wells) or microscope slides. The sorts can be done in aseptic conditions and the cells are suitable for culture. In addition, the Facility runs 1 Becton-Dickinson LSRII for analysis with 5 lasers so that users can perform 10-colour analyses. The acquisition of another FACS analyzer is also being considered to meet the increasing demand of users.

Key Operator
Michelle Mok micmok@nus.edu.sg 12N Bench 11

We are able to answer all enquiries on:

  • Sample preparation requirements
  • Instrument configuration and set-up (including custom filter configuration)
  • Suitability of fluorochrome combinations
  • Application support
    • Compensation
    • Cell sorting
    • Immunophenotyping / Cell signaling
    • Cell cycle analysis
    • Apoptosis Assays (Annexin V, Caspase, Cyanine dyes etc)
    • Cell Proliferation (BrdU, EdU, PKH26, CFSE)
    • Stem Cell Side Population
    • Calcium Flux
    • Reactive Oxygen Species (ROS)
    • Drug Uptake / Phagocytosis
  • FACSDiva and FlowJo software

We have FACSDiva (for Windows XP) and FlowJo (for Macintosh/ Windows) dongles available for lease (3 hours). The dongles will allow you to install and analyse your data on your own computer. Please contact Michelle.

CSI FACS Facility Access Policy

Operation Hours

  • 8:30am to 9pm (Monday – Friday, excluding public holiday)
  • Operator-assisted FACS Aria sorting service is available between 9am to 9pm.
  • The FACSAria cell sorter can be used 24 hours a day, 7 days a week. However, self-operated sorting is expected on weekends & after 9pm on weekdays and is opened to trained staff only.
  • Self-operation is required for analysis by LSRII at all times. We can give you a basic walkthrough if you are a first time user.

 

Booking System

  • Our booking website is –> https://csi.nus.edu.sg/facs/
  • To register for an account, please contact us at csifacs@nus.edu.sg
  • Booking is required for use of the FACS Aria & LSRII at all times, including usage during non-chargeable hours.
  • Minimun notice period for cell sorting is 12 hours (office hour timeslots). Cell sorting appointments from 6pm to 9pm must be made minimally one day in advance. Please notify us via EMAIL for urgent evening access to the cell sorter.
  • Maximum booking hours for LSRII is 3 hours.
  • User will be notified after booking is approved.
  • All usage of the LSRII PC is chargeable, even for “Data Analysis” use only. Users are encouraged to borrow our software dongles to perform data analysis on their own PCs.
    (Please contact us for more information on charges)
  • Charging takes into effect from the time service is booked, regardless of your punctuality. Charging ends according to the time booked on the system, or the actual end time of the service, whichever occurs at a later hour.
  • No service charge during non-facility operation hours.
  • Small packets of 5ml FACS tubes (25 tubes) is available for sale to users who will be running their samples at CSI FACS.
  • Please contact us (Edward Shi: csisj@nus.edu.sg) for further information and price list.

 

Cancellation
Cancellation notice received > 2 days before day of service No penalty
Cancellation notice received 0 – 2 days before day of service 100% service charge

Cancellation can be made by deleting the booking from the online system.