Functional studies of gene variants

Question 1

2 strategies to express gain of function

Answer 1

1. over-expressing the mutant protein
2. Keeping allelic balance as it is a dominant transmission

Question 2

What model is used to study gain-of-function?

Answer 2

Knock-in model: transgenic mouse

Question 3

Which models are used to study loss-of function?

Answer 3

Knockout and knockdown gene expressions

Question 4

What is knockout gene expression?

Answer 4

You get rid of the gene–> eliminate its expression

Question 5

What is knockdown gene expression?

Answer 5

downregulating the gene’s expression significantly

Question 6

What should not be modelled using a knockout gene?

Answer 6

Dominant negative mutation

Question 7

Cell models that can be used

Answer 7

1. Immortalised cell lines
2. Primary cells isolated from the tissue of interest
3. Induced pluripotent stem cells
4. Organoids

Question 8

What are organoids?

Answer 8

3d structures that resemble the organ of origin that are derived from stem cells which are then differentiated

Question 9

RNA interference

Answer 9

KNOCK DOWN: a technique that uses siRNAs to target a specific RNA and silence its expression

Question 10

Describe the process of RNA interference

Answer 10

SiRNA binds to complementary sequences of target mRNA which triggers the degradation of mRNA or the inhibition of its translation into a protein

Question 11

How can a cell repair a double-strand break caused by CRISPR/cas9 knockout?

Answer 11

1. Homology directed repair
2. Non-homologous end joining NHEJ

Question 12

Homology directed repair

Answer 12

Uses homologous recombination to synthesise the missing piece so that the precise changes or insertions can be introduced at the site of the break

Question 13

NHEJ

Answer 13

error prone: features giving together the 2 strands of DNA without a template

Question 14

What does gene targeting use?

Answer 14

Homologous recombination

Question 15

Process of gene targeting

Answer 15

1. Isolate embryonic stem cells from blastocysts and we can culture them in vitro
2. The cells are placed in the presence of a targeting vector
3. The plasmid will match and induce homologous recombination between the target sequence and plasmid causing an exchange of genetic information
4. Cells that were successfully modified are injected into a blastocyst to create a chimeric blastocyst creating chimeric offspring
5. The chimeric mouse is crossed with a wt mouse to obtain a heterozygous mutant mouse

Question 16

Chimeric offspring

Answer 16

An organism containing cells derived from 2 or more genetically identical zygotes

Question 17

What is used for positive selection?

Answer 17

Neomycin an antibiotic

Question 18

What is used for negative selection?

Answer 18

Thymidine kinase
When we give ganciclovar to the cells, only the cells that express Tk will die

Question 19

What is conditional inactivation?

Answer 19

Knock out a gene present in certain tissues

Question 20

loxP system

Answer 20

1. the target gene we want to eliminate is flanked with loxP sites: when recombination is induced, the gene between the 2 sites will be cut out
2. The mouse is crossed with another transgenic mouse in which a tissue-specific promoter linked to a Cre gene is introduced
3. The complex induces recombination between the 2 loxP sites which will be present in all cells of the body but the Cre enzyme will be present only in certain cells
4. Their offspring will have the Cre transgene allowing the target gene to be eliminated in those specific tissues

Question 21

What is another technique with similar principle as Cre?

Answer 21

Flippase (FRT)

Question 22

What does it mean when we have the presence of the enzyme Cre?

Answer 22

it induces recombination

Question 23

Inducible inactivation

Answer 23

Deletion of a gene only in certain tissues at a certain time

Question 24

What is the principle of inducible inactivation?

Answer 24

Estrogen receptors are present in a cell in the inactive state and they are bound to HSP90, a chaperone that keeps the protein in the cell inactive. When we give estrogen to cells they bind to the estrogen receptor and upon this binding HSP90 releases this receptor.

Question 25

What are the 2 methods to make Cre inactive?

Answer 25

1. Fusing the Cre protein with a part of the estrogen receptor
2. Tetracyclin resistance operon

Question 26

Fusing the Cre protein with a part of the estrogen receptor

Answer 26

Estrogen receptor has been modified to bind an estrogen-like molecule instead of estrogen. Tamoxifen causes the release of HSP90 from teh estrogen receptor so the Cre protein becomes active. Cre will go to the nucleus and catalyse the recombination

Question 27

Tet-off system

Answer 27

Tetracyclin/ Dox is sued to switch off the expression of the gene driven by Cre and activated by TTA protein

Question 28

Tet-on system

Answer 28

rTTA is designed to let the system work in the other direction: when we give Dox cyclin, it binds to rTTA which binds to tetO and activates expression of CRe

Question 29

What happens if you use CRISPR cas9 at the 2-cell or 4-cell stage?

Answer 29

Then part of the cells be edited and the others will be wt creating a mosaic

Question 30

Transduction

Answer 30

Using a lentiviral/adenoviral vector to stably introduce the expression of a gene of interest within a cell

Question 31

Transfection

Answer 31

Using an expression vector that contains a cDNA of the gene that we are interested in: can involve a nucleic acid sequence such as siRNA/ miRNA or a plasmid

Question 32

What happens if a nucleic acid sequence is used in transfection?

Answer 32

The sequence will remain at the cytosolic level

Question 33

What happens if a plasmid is used in transfection?

Answer 33

It may be introduced in the nucleus and integration within the DNA. Antibiotics are given so that only the cells with plasma integrated within their DNA will survive

Question 34

Stable transfection

Answer 34

Transfection selected for integration

Question 35

Transient transfection

Answer 35

Acute phase [short period of time]

Question 36

Stable transfection vs transient transfection

Answer 36

1. DNA vectors are used only; DNA vectors and RNA can be used
2. integrates in the genome; not integrated in the genome
3. passed onto progeny; not passed onto progeny

Question 37

Hemizygous

Answer 37

has one chromosome carrying the transgene and one chromosome not carrying the trans gene

Question 38

What should our control mouse be if we have overexpression of a mutant?

Answer 38

Our control mouse should have overexpression of out wt protein so that the overall level of expression can be comparable

Question 39

What can avoid the trans gene from the regulation of chromatin?

Answer 39

Insertion of insulator sequences