Introduction

  • Three steps for approval of project involving animals. Each step takes approx. 1 month.

    graph LR Pre-submission --> ORBEA-->DGAV

  • ORBEA: Animal welfare body

  • DGAV: Portugese government organisation that licences facilities.

  • They have 50 year old preserved embryos.

  • Plant Model organism: Nicotiana benthamiana.

  • Gnotobiology comprises the study of germfree plants and animals, as well as living things in which specific microorganisms, added by experimental methods, are known to be present.

  • Metabolic cage: readout-rate is about 1 Hz. Food, water intake; body weight; $O_2$ intake, $CO_2$ output, etc are tracked.

Transgenic Units

Mice Facility

  • Embryonic stem cells (ESCs) are found in the inner cell mass of the human blastocyst, an early stage of the developing embryo lasting from the 4th to 7th day after fertilization. In normal embryonic development, they disappear after the 7th day, and begin to form the three embryonic tissue layers.
  • Gene insertion:
    • A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another.
    • linear BAC vectors (can harbour upto 350kb gene sequence) — more specific insertion of the gene in the host embroyo genome.

BE $\mu$FAB

3D Printing:

  • Material Extrusion: Fused Filament Fabrication (FFF)

  • Photopolymerisation Stereolithography (SLA) — Resin Printer

  • PEEK (Polyether ether ketone) — very resistant to heat and chemical erosion.

  • OpenSCAD: Has a freely integrated terminal interface.

  • Specific Bed and Nozle temperatures are defined for different materials.

Microfluidics

Flow Control Systems

  • Peristatic Pumps: sets up large flow rates. The flow rates are not constant due to constant work on the tube.
  • Syringe Pumps: Recommended use with glass syrenges which have Teflon plungers.
  • Pressure driven pumps: No oscillation flow. 0.005% stability. Pressure limited to 8 bars. ~20-30k€.
  • Valves: injector, selector, and matrices.

Flowcytometry

  • The cells must be in suspension for flow cytometry measurement.

  • Scale of measurement: 200nm (Nano-scale Cytometry) to 20-30$\mu$m.

  • Forward Scatter measures the size of the particle using Diffusion Light Scattering.

  • Side Scatter measures the granularity of the cells.

  • What kind of detectors/filters is used in FC Cytometry? — An array of filters.

  • Sorting: ~4% sorting error and 4-way sorting is available at IGC.

  • TODO: There are dyes that intercalate the DNA: ethidium bromide.

Advanced Imaging

  • PPBI: Portugese plateform for bioimaging & EuroBioImaging.
  • Phase contrast microscopy: Fritz Zernike received the Nobel Prize for it. Zeiss commercialised it.
  • NA: Numerical Aperature
    • High NA has a higher resolution.

Electron Microscopy

  • Aldehydes — Glutaraldehyde and formaldehyde are used in a cocktail mix. Formaldehyde is a reversible fixative. With Glutaraldehyde, the fixation is permanent but slow.
graph TD; 1(Primary fixation
with Aldehydes) -.-> bw[[buffer-wash]] -.-> 2(Secondary fixation
with Osmium Tetroxide) 2 -.-> ww1[[water-wash]] -.-> 3(Add electron density
wityh Uranyl Acetate) 3 -.->ww2[[water-wash]] -.-> 4(Dehydration series with ...) 4 --> 5(xxx)
  • CLEM: Correlative Light and Electron Microscopy
    • Concurrent Light and Electon microscopy.
    • Two protocols: Pre-embedding and Post-embedding.
  • Five kinds of EM:
    1. Serial Section TEM
    2. Serial TEM Tomography
    3. Array Tomography
    4. Serial Block Face SEM
    5. Focused Ion Beam SEM

Genomics

  • Whole Genome Sequencing Cost: ~250,000$

  • Transcriptomic reads are done by reading the RNA.