Techniques

    • Transgenesis in mice. CRISPR and classical techniques.
    • Microinjection into post-implantation mouse embryos.
    • Electroporation of embryos with CRISPR
    • Microinjection of post-implantation embryos (Cre-Recombinase)
    • Management of tetraploids (obtaining, aggregations and microinjection)
    • Enucleation / Electrofusion techniques with karyoplasts and cytoplasts for the generation of conplastic mouse lines
    • Pre-implantation mouse embryo culture
    • Experimental embryology of birds
    • Comparative genomics: bioinformatic and evolutionary analysis of genomes
    • General techniques of Molecular Biology
    • Germplasm Cryopreservation Techniques: embryos and sperm
    • Ovariectomy
    • Ovarian transplant
    • Artificial insemination techniques: intrabursal, transoviductal and intrauterine
    • Cryopreservation Techniques for Zebrafish germplasm: sperm.

    Equipment

    a) Microinjection Laboratory:

    • 1 NUAIRE (US AUTOFLOW CO2 Water Jacketed Incubator) CO2 Incubator Model UN-4750 maintained at 37°C and 5% CO2 (in air) for culture of mouse pre-implantation embryos
    • 1 Sutter Model P-97 automatic micropipette puller (Flaming/Brown Type Micropipette Puller) for manufacture of micropipettes
    • 1 DeFonbrune microforge (“The GlasswoRx” Model ICSI F-1200-2) with a Olympus compound microscope fitted with 10x lenses (F-5 micrometric/goniometric reticulum in one) and 4x and 10x objectives. Used for manufacturing glass microtools
    • 2 Leica stereoscopic microscopes. One is a Model MZ-10F (with built-in fluorescent source as well as digital camera and TFT monitor for simultaneous viewing and scientific documentation, and a peripheral computer system connected to the center’s intranet). The other is a Model MZ-12.5. Both stereomicroscopes have diascopic bases, and are used for the collection and management of mouse oocytes and pre-implantation stage embryos and cells in suspension in general
    • 1 Nikon Model SMZ1000 stereoscope with a diascopic base for the collection and management of mouse oocytes and pre-implantation stage embryos
    • 2 LEICA Model CLS150X light sources with cold light with two semi-rigid fiber optic cables (Incident light AL/S 4.5 mm diameter 2x450mm/750) and SH2E 4.5 mm/600 mm flexible two-arm fiber optic cable
    • 2 ZIESS AxiObserver inverted microscopes (both with digital camera and TFT monitor for simultaneous viewing and scientific documentation, and a peripheral computer system connected to the center’s intranet). These microscopes incorporate Nomarski optics (DIC, Differential Interferential Contrast) to visualize cells in suspension and mouse pre-implantation embryos destined for genetic modification. They have 10x lenses and 5X, 10X, 20X (DIC) and 40X (DIC) objectives
    • 1 LEICA Model DMIREZ inverted optical microscope with 10X lenses and 5X, 10X, 20X (DIC) and 40X (DIC) objectives to visualize cells in suspension and mouse pre-implantation embryos destined for genetic modification
    • 6 Eppendorf TransferMan-NK2 motorized micromanipulators (2 for each microscope) which allow controlled and precise movements required for microinjection of mouse embryos
    • 1 Eppendorf CellTram-Air manual microinjector for holding mouse oocytes and embryos
    • 2 Eppendorf CellTram-Vario hydraulic microinjectors with gear assembly for injection of genetically modified ES-Cells into E8C or blastocysts to generate chimeric mice and produce KO or KI mice or for ICSI of mouse oocytes
    • 3 Eppendorf CellTram-Oil hydraulic microinjectors for holding mouse oocytes and embryos
    • 3 Eppendorf FemtoJet digital pneumatic microinjectors, one for each microscope, for microinjection of pronuclei with DNA solution and for subzone microinjection with recombinant lentivirus
    • 2 PrimeTech Model PMM-150-FU piezoelectric microperforators
    • 3 TMC Series 63-500 anti-vibration tables (750 x 1200 mm dimensions) with active supports. This is where the microinjection equipment is mounted
    • 1 Advanced Instruments Inc. Osmometer, Model 3320
    • 1 Laser for embryo micromanipulation. Model XYClone brand Hamilton Thorne
    • 1 Electroporator

    b) Embryo Transfer Laboratory:

    • 1 REVCO Model MRI3000S-9-VBA CO2 Incubator maintained at 37°C and 5% CO2 (in air) for culture of mouse pre-implantation embryos
    • 1 BUNSEN Model 1530 digital plate heater for heating the containment cage (to 37°C) used to house female mice that have undergone surgery for embryo transfer
    • 2 Leica Model MZ-12.5 stereoscopic microscopes (one with a digital camera and TFT monitor for simultaneous viewing and scientific documentation, and a peripheral computer system) for collection and handling of mouse pre-implantation embryos for embryo transfers
    • 2 Keller Steri-250 glass bead sterilizers to sterilize surgical instruments
    • 2 LEICA Model CLS150X light sources with cold light with two semi-rigid fiber optic cables (Incident light AL/S 4.5 mm diameter 2x450mm/750) and SH2E 4.5 mm/600 mm flexible two-arm fiber optic cable
    • 2 temperature controllers (F.S.T. TR-200) with rectal probe and heated pad for mice. Used to prevent hypothermia in mice undergoing surgery