TS 1 Flashcards
In Canada how is animal use in teaching and research governed?
Through the Canadian Council on Animal Care
What is the CCAC?
A non-profit organization to enact high standards of animal ethics and care in science throughout Canada
What are institutions funded through tri-council (NSERC, CIHR, SSHRC) required to obtain?
A certificate of Good Animal Practice from CCAC
When are animals in research, testing, and teaching acceptable?
Only if it promises to contribute to the understanding of fundamental biological principles or to knowledge that can benefit humans/animals
When should animals be used?
Only if the researcher’s best efforts to find an alternative have failed
What are researchers with animals required to do?
Employ the most human techniques and use the smallest number of animals
What is required for the institution to get certification?
- Every 3years a panel of scientists, veternarians, and community members from CCAC visit UofC
- The panel identifies strengths and weakness and makes suggestions to improve
- The UofC must submit a response explaining how they would address the changes from CCAC
Who needs to approve ALL animal care use at the UofC?
Animal Care Committee
What does ACC look at?
- Obsersvational vs. experimental
- Wildlife vs. lab animals
- Researchers vs. teachings
What does the reporting and training involve?
- Annual updates detailing the number/type of animals used must be submitted
- Animal use protocols must be re-reviewed every 4 years if they are still in-use
- All users handling either live animals or tissues at the UofC must complete the IAUTP module
What do the ranking of categories represent?
The level of invasiveness
What is level A?
Tissues/blood COI such as this class
What is level B?
Experiments which cause little or no discomfort to the animals
What is level C?
Experiments which cause minor stress or pain for short durations
What is level D?
Experiments which cause moderate to severe distress or discomfort such as Dr. Cobb’s lab
What is level E?
Procedures which cause severe pain, near, at or above the pain tolerance threshold of unasthetized/conscious animals
What are the 3 R’s?
Replace
Reduce
Refine
What is replace?
Having alternative to animal use
What is reduce?
Using the fewest number of animals
What is refine?
Have activities using animals that have been optimized to reduce distress
Why does location matter?
The gene’s expression location can tell us the function as well as its relationship with other genes
What is SHH?
Is a ligand
What is PTCH1?
Is a receptor
What is Bmp7?
The gene that is expressed in the webbed hands between the digits and leads to apoptosis
What is the process of SHH binding?
- SHH is transcribed by a cell containing the gene and is then translated to make the SHH protein
- SHH protein (ligand) that binds to the PTCH1 receptor
What is WISH?
Whole-mount In Situ Hybridization which involves staining the whole embryos and tells us where in an embryo a given gene is transcribed or expressed also it uses a complementary RNA probe
What is the WISH probe?
A complementary RNA probe that will hybridize to the template mRNA sequence in the embryonic cell and it is the complementary dioxigenin (DIG)-labeled RNA probe
How is the complementary dioxigenin (DIG)-labeled RNA probe made?
In a test tube by in vitro transcription
What is added to the test tube?
- Template target gene DNA
- Nucleotides A, G, C and U labeled with DIG label
- RNA polymerase
What happens where there is a U or a uracil?
DIG will bind and will form a large bulky compound
What is the process for the WISH assay?
- Add DIG-labeled probe to fixed embryos
- Corresponding to the gene the probe hybridizes to the complementary mRNA
- Detect the probe with the anti-DIG antibody attached conjugated to an enzyme
- Enzyme conjugate turns added substrate purple to reveal where and (when) the gene is expressed
What is the anti-DIG antibody?
Alkaline phosphatase
What is the reporter gene?
When this gene is expressed it makes a product that you can detect
Where are the places that the reporter gene can be expressed?
- Along with gene of interest
- In place of gene of interest
How are reporter genes engineered?
Through homologous recombination
What is LacZ?
A reporter gene that encodes for B-galactosidase
What does B-galactosidase do?
It carries out a reaction to stain the embryo when the substrate is added
What does the location of staining indicate?
Where the gene is expressed and its function
What happens when a reporter gene is inserted into the coding sequence of a gene?
It nullifies it
What happens when a reporter gene is inserted in-line with the coding sequence of a gene?
If it does not disrupt the reading frame then it is translated with an internal ribosome entry site
What happens if it attached to the coding sequence of a gene?
It creates a fusion protein
What are enhancers?
These are known DNA sequences that are cell, tissue, and stage-specific and promote transcription activity by binding transcription factors and each enhancer has different strengths
What is a transgenic reporter assay?
It uses a reporter gene to show when the enhancer is active
What is introduced into an embryo to make a transgenic animal?
- DNA construct that contains the enhancer sequence
- A promoter
- A reporter gene
How is the enhancer related to the reporter gene?
It expresses the reporter gene
What does the reporter gene expression correspond to?
When and where the enhancer is active
the location of staining => where the enhancer activated LacZ
What is a pronuclear injection?
This is a method of making a transgenic animal
What is the process for pronuclear injections?
Use a thin glass pippette to inject the linear DNA into the pronucleus of a fertilized egg
What is a pronucleus?
This is when the maternal (egg) and paternal (sperm) DNA in the pronuclei are not fused
What happens when the linear DNA is randomly incorporated?
It can be inserted into an essential gene and kill the embryo or a reporter gene may be activated and lead to abnormal expression somewhere else
What happens after the injection?
The pronuclei eventually fuse to create a single nucleus for the zygote
How many contstructs are incorporated?
Multiple copies
What is the luciferase assay?
This determines if the sequence has enhancer activity or not and quantifies its strength
What is a minimal promoter?
Allows for the formation of the initiation complex between the coding sequence and the enhancer
What is the enhancer of interest added to?
Its inserted into a vector that has a minimal promoter and the luciferase reporter gene
How is the vector added to cells?
The vector with the enhancer of interest is transfected into cells
What will the enhancer do?
Drive luciferase expression which will be quantified by a luminometer
How can we determine the strength of an enhancer and quantify how it changes?
- Alter the nucleotide sequence of the enhancer and disrupt the transcription factor binding sites
- Add or remove transcription factors
What is a loss of function mutation?
Knockout of a gene
What is a gain of function mutation?
Overexpression of a gene
What is endogneous expression?
Where the gene is normally expressed
What is ectopic expression?
Expression of a gene where it is not normally found
What is standard gene knockout?
Disrupting the DNA sequence in the gene of all the cells of the organism so it is no longer able to make a protein
What causes a standard gene knockout?
A DNA construct
What is homologous recombination?
Where similar DNA sequences are exchanged between the construct and the endogenous gene
What is used as a marker in the construct?
A gene that encodes for antibiotic resistance
What happens after the cells with resistance are selected?
- The cells are injected in the inner cell mass of the blastocyst
- The blastocysts are then implanted into a surrogate and grown
- Chimaras are bred
What is the difference between a transgenic mouse and knockout mouse?
Transgenic:
1. Edits all the cells in the organism
2. Makes a chimara
What is conditional gene knockout?
If you conduct a standard gene knockout on an essential gene the organism dies
What is cre?
A viral enzyme that excises genes that are floxed
How do you flox a gene?
Surround it with LoxP sites on either sides
Does floxing a gene disrupt its function?
No
How is cre expression driven?
With cell specific promoter and enhancers
What happens wherever the cre is expressed?
The floxed genes are removed
What is a constituitive promoter?
It drives gene expression in all cells constantly
How can we use this Rosa26 promoter for a gain of function experiment?
Have an extra copy of the gene be expressed
What is a stop cassette?
Any region of the DNA sequence with labelled with LoxP sites between the promoter and the target gene
What does the stop cassette allow?
The gene can be conditionally overexpressed because cre will excise just the floxed region
