How do we study cells?

Question 1

how can we genetically manipulate gene expression in cells?

Answer 1

Take out genes

Add genes in

Question 2

How can we determine the importance of a gene?

Answer 2

Remove the genes expression and study the effects

Question 3

What are ways to decrease gene expression from a cell?

Answer 3

RNA interference (siRNA / RNAi)

Genetic knocknout mice

CRISPR gene editing

Question 4

How does siRNA work?

Answer 4

1. DICER molecule cleaves part of the dsRNA after dsRNA processing
2. RISC binds to the dsRNA and cause unwinding of the dsRNA
3. RISC is then recruited to the mRNA target molecule.
4. The target mRNA is cleaved and the gene is removed.

Question 5

What is RISC?

Answer 5

RNA induced silencing complex.

Question 6

What are the main genetic knockout methods in mice?

Answer 6

Non-conditional knockout - all cells in mouse will be deleted for the protein

Conditional knockout - tissue specific gene deletion

Question 7

What is the problem with non-conditional knockout mice?

Answer 7

Can cause embryonic lethality

Question 8

How are conditional knockout mice made?

Answer 8

Using the Cre-loxP system where Cre recombinase will remove the gene from a specific tissue

Question 9

How does CRISPR gene editing work?

Answer 9

Cas9 is a bacterial nuclease that targets DNA.
Guide RNA is complementary to that target DNA sequence
Together they enable specific targeting to DNA and its cleavage
Insertion of a mutated donor DNA sequence allows for targeted genome editing.

Question 10

What experimental techniques are used to overexpress a protein in a cell?

Answer 10

Transfecting in DNA that encodes for the protein

Transgenic overexpression in mice.

Question 11

Why do we look at size and shape of cells?

Answer 11

Using microscopes they can reveal specific traits

eg. elongated cells may be more invasive

Question 12

What method is useful for detecting proteins?

Answer 12

Using antibodies coupled to fluorophores allows us to visualise DNA damage.
Targeting proteins to GFP for cellular localisation

Question 13

How do antibodies detect the proteins?

Answer 13

Protein X
Primary antibody joins
Secondary antibody joins to primary (conjoined with fluorophore)
excitation with one wavelength causes emission of light.

Question 14

How do we use proteins as cellular markers or compartments?

Answer 14

Different proteins can be tagged to show their localisation within the cell.
Eg. Histone proteins will be localised to the nucleus.

Question 15

How are antibodies used in protein biochemistry?

Answer 15

Coupling antibody to enzyme HRP (horse radish peroxidase)
HRP will produce a light signal due to being chemilluminescent
Carry out western blotting to detect protein concs based on light intensity emitted.
Use western blot to detect protein PMTs

Question 16

How can separate parts of protein complexes be identified?

Answer 16

Using mass spectrometry

Question 17

How are antibodies used in pathology?

Answer 17

Used via immunohistochemistry

they can be used a drugs for clinical diagnosis (herceptin)