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. Following services are presently offered by the Transgenic & Gene Targeting Facility:
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.
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).
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.
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.
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.
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.
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)