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 & 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. The transgenic facility provides services and collaborative supports to generate genetically-engineered mouse models for basic and translational biomedical research in academic organizations and biotech industries.

Services

Genome editing in mice by CRISPR/Cas 9 technology (New)

We have successfully used CRISPR/Cas9 technology to create targeted genome mutation, editing and DNA cassette knock-in into pronuclear mouse and rat embryos to generate engineered animal models. This service now available to PIs/Scientists in NUS and other research /academic Institutes to generate animal model with their preferable animal strains (e.g., FVB/N or C57BL/6).

Collaboration for knock-out & knock-in mice (New)

Transgenic & Gene Targeting Facility invites for collaborations for the generation of knock?out or knock?in mice for any locus of interest. We also invite collaboration for the generation of Rosa26 based targeted transgenic mice.  We accept 8?12 projects in a year from PIs in academic institutes in order to ensure low cost and effective time management for high impact research goal. We offer collaborations starting from vector design/construction to establishment of targeted heterozygous mice for your gene of interest in 9-12 months time.

Generation of transgenic mice by pronuclear DNA injection

Please prepare your DNA according to the guideline of facility and submit your DNA construct (at least 10 ug at a concentration of 0.1-0.2 ug/ul). We strongly recommend you to remove traces of vector base from the construct and we guarantee 3 founder transgenic mice for neutral construct or injection of 200 zygotes (at post-injection survival rate of 50-80%) of FVB/N or C57BL/6 strains. PI should be responsible for genotyping to confirm transgenic founders.

Gene targeting in embryonic stem (ES) cells

  1. Performance validation of targeting vector: Please submit construct map and circularized plasmid construct (at least 500 ug at concentration of 0.5-1.0 ug/ul). Please also indicate unique restriction site for linearization and selection markers in the construct map. We will optimize ES cell culture and positive-negative selection conditions for your construct. Performance of your vector in colony generation against positive-negative selection will be determined and compared with our in-house control vector. Acceptable vector performance should be above (1: 20.000), i.e., generation of a single colony out of twenty thousand ES cells elcctroporated following our standard specified condition.
  2. Embryonic stem (ES) cell culture and manipulation for gene targeting: Currently we offer gene targeting in ES cells derived from 129Sv/Ev mouse strain. We will electroporate your validated targeting vector. After the completion of positive-negative selection, 96-288 ES cell clones will be isolated for primary screening and freezing in 96-well format. PI will be responsible for the primary screening homologous recombination in isolated ES clones.
  3. Primary screening of ES clones by long-PCR: We encourage PIs to establish this assay for the identification of homologous recombination in ES cell clones in their own lab. However, we offer this service to PIs who don’t have appropriate manpower or experience in this assay. Please submit details of vector construction, map and Gene bank accession number. A long-PCR based pseudo-screening strategy will be established for the detection of homologous recombination in ES clones. We will ensure the detection of homologous recombination at least in three independent clones or completion of PCR-screening of 288 (3×96) clones. (Note: should pseudo-screening strategy can’t be established, a lump sum charge, will be applied and PI should be responsible for the screening of clones by alternative methods, e.g., Southern blotting hybridization technique).
  4. Genotyping of ES clone by Southern blotting & DNA hybridization: Establishment of Southern blotting & DNA hybridization procedures for the copy number detection of targeted allele is required for the secondary confirmation of homologous recombination in ES clones. This service is not currently offered by the transgenic facility. PI will be responsible for the secondary screening of homologous recombination. A guideline and a general standard protocol (SOP) will be supplied by the transgenic facility. We strongly recommend PIs to establish this assay in their own lab before submitting targeting vector or ES clone for transgenic services.

Expansion of ES cell clones

We provide service to expand ES clones for cryo-preservation, secondary screening and microinjection. Upon expansion, genotyping of ES clones should be further confirmed by the PI.

Generation of chimera mice from targeted ES cell clones

Please submit 2-3 independent targeted ES clones with proper documentation and certification for the source and characterization, i.e., genotyping, mycoplasma and MAP (Mouse Antibody Production) test status of ES cells. We will ensure ES cell injection into 100-150 blastocysts with a survival rate of 70-80% and subsequent uterine transfer of survived blastocysts for chimera mice production.

Germ-line rescue & cryo-preservation

The genetically modified mice generated through transgenesis either in mouse embryos or in embryonic stem (ES) cells are powerful tools for studying gene function and regulation. These models serve as invaluable resource for preclinical study for therapeutic and diagnostic target discovery and validations. However, in some occasions it can be difficult to successfully breed and maintain transgenic animals for long term. To overcome these problems, investigators have focused on the development and refinement of Assisted Reproductive Techniques (ART) and Cryogenic Techniques (CT) over the last decades. At present, ART and CT strategies are proven to be instrumental to rescue, maintain and archival of transgenic lines for biomedical research and development. We offer service using some of the following techniques:

  1. Ovary transplantation, to rescue mouse line from female breeder that stops breeding- upcoming
  2. Embryo transfer, to rescue mouse lines free from infections.
  3. In-vitro fertilization (IVF), to rescue mouse line from male breeder that stops breeding.
  4. Intracytoplasmic sperm injection (ICSI), to rescue mouse line from male breeder with low sperm count or sperm motility defects- upcoming
  5. Tetraploid embryo complementation, to establish mouse line through ES cell technology, when heterozygous manipulation causes infertility- upcoming
  6. Vitrification for mouse embryo and sperm cryopreservation.

(Note: Please contact us for your need and interest in these ART & CT approaches for re-derivation of mouse line and cryo-preservation mouse embryos and sperm)

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.

 

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.