Tutorial - Quiz 1 Flashcards
What are the conventional model organisms
Mice, Zebrafish, Chicks, Drosophila, C. Elegans, Xenopus, axolotol, organoids
In Canada what is animal use covered by
The Canadian council on animal care (ccac)
All animals care and use atr uofc is approved by
Animal care committee (ACC)
3 things they have to report
Number and type of animal
Protocol reviewed every 4 years
Complete training
The three rs are
Replace: Have alternatives to vertebrate animal use been considered (cell culture, invertebrates)?
* Reduce: How are the fewest number of animals necessary being used?
* Refine: Have activities involving animals been optimized to reduce distress as much as possible?
Finding where a gene is expressed is a starting point to figure out what
where it functions, what its functions are, it’s relationship with other genes
WISH stands for
whole-mount In situ Hybridization
What does wish use?
It uses a complementary RNA probe
What does WISH involve?
staining the entire embryo to tell where a given gene is transcribed or expresses
A complimentary RNA probe will be
hybridize to transcribed sequences in the cells of an embryo
This complimentary — is made in a test tube by what
This complimentary digoxigenin (DIG) - label RNA probe is made in a test tube by in vitro transcription
The steps of WISH Assay are
- Add DIG-labeled prob to fixed embryos (the probe will hybridize to complementary mRNA corresponding to a gene)
- The you detect the probe with an anti-DIG antibody which is attached to an enzyme
- The enzyme conjugate turns added substrate purple, revealing where and when a gene is expressed.
what makes a product that’s detectable
Reporter genes
Reporter genes are engineered into the genome using
homologous recomb
The Lacz reporter gen encodes
the b-galactosidase enzyme
Reporter genes can be…
Inserted into coding sequences, nullifying it
Inserted in-line with the coding sequence of a gene without disrupting the reading frame (with and internal ribosome entry site)
Attached to the coding sequence if a gene (making a fusion protein)
What is an enhancer?
DNA sequences that communicate with a gene promoter to activate transcription for that gene
So they contain…
Binding sites for trancsription factors
Eukaryotic enhancers have diff
“strenghts” so the amount of trancription that happens is variable
Transgenic reporter assay use
reporter genes to show when and where a enhancer is active
What is used to make a transgenic animal?
A dna construct contianing an enhancer, promoter and the reporter gene in an embryo, so where u get staining is where the enhancer is active obvi
How do they generate transgenic animals?
A thin glass pipette and a small volume of linear DNA is injected into the a of a fertilized egg
Once injected the linear dna is
randomly seperated betweeen the pronuclei and then they eventually fuse ti make a single nucleus fore the zygots
What is luciferase assay used?
To determine wether a certain sequence has enhancer activity or not and ALSO to quantify enhancer activity
So how does luciferase work?
An enhancer of interest is inserted in a vector that has a minimal promoter and the luciferase promoter gene. The vector is transfected into cells and then the enhancer activity can be quantified by a luminometer (how much it glows
What are the two ways you can determine the strength of an enhance/ quantify how it changes
Altering the nucleotide sequence of the enhancer, Add or remove transcription factors
