Tutorial - Quiz 1

Question 1

What are the conventional model organisms

Answer 1

Mice, Zebrafish, Chicks, Drosophila, C. Elegans, Xenopus, axolotol, organoids

Question 2

In Canada what is animal use covered by

Answer 2

The Canadian council on animal care (ccac)

Question 3

All animals care and use atr uofc is approved by

Answer 3

Animal care committee (ACC)

Question 4

3 things they have to report

Answer 4

Number and type of animal
Protocol reviewed every 4 years
Complete training

Question 4

The three rs are

Answer 4

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?

Question 4

Finding where a gene is expressed is a starting point to figure out what

Answer 4

where it functions, what its functions are, it’s relationship with other genes

Question 4

WISH stands for

Answer 4

whole-mount In situ Hybridization

Question 5

What does wish use?

Answer 5

It uses a complementary RNA probe

Question 5

What does WISH involve?

Answer 5

staining the entire embryo to tell where a given gene is transcribed or expresses

Question 5

A complimentary RNA probe will be

Answer 5

hybridize to transcribed sequences in the cells of an embryo

Question 6

This complimentary — is made in a test tube by what

Answer 6

This complimentary digoxigenin (DIG) - label RNA probe is made in a test tube by in vitro transcription

Question 7

The steps of WISH Assay are

Answer 7

1. Add DIG-labeled prob to fixed embryos (the probe will hybridize to complementary mRNA corresponding to a gene)
2. The you detect the probe with an anti-DIG antibody which is attached to an enzyme
3. The enzyme conjugate turns added substrate purple, revealing where and when a gene is expressed.

Question 8

what makes a product that’s detectable

Answer 8

Reporter genes

Question 9

Reporter genes are engineered into the genome using

Answer 9

homologous recomb

Question 10

The Lacz reporter gen encodes

Answer 10

the b-galactosidase enzyme

Question 11

Reporter genes can be…

Answer 11

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)

Question 12

What is an enhancer?

Answer 12

DNA sequences that communicate with a gene promoter to activate transcription for that gene

Question 13

So they contain…

Answer 13

Binding sites for trancsription factors

Question 14

Eukaryotic enhancers have diff

Answer 14

“strenghts” so the amount of trancription that happens is variable

Question 15

Transgenic reporter assay use

Answer 15

reporter genes to show when and where a enhancer is active

Question 16

What is used to make a transgenic animal?

Answer 16

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

Question 17

How do they generate transgenic animals?

Answer 17

A thin glass pipette and a small volume of linear DNA is injected into the pronuclei of a fertilized egg

Question 18

Once injected the linear dna is

Answer 18

randomly seperated betweeen the pronuclei and then they eventually fuse ti make a single nucleus fore the zygots

Question 19

What is luciferase assay used?

Answer 19

To determine wether a certain sequence has enhancer activity or not and ALSO to quantify enhancer activity

Question 20

So how does luciferase work?

Answer 20

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

Question 21

What are the two ways you can determine the strength of an enhance/ quantify how it changes

Answer 21

Altering the nucleotide sequence of the enhancer, Add or remove transcription factors