Studying Gene Expression 1, 2 & 3

Question 1

Give the steps of using mRNA to study differential gene expression?

Answer 1

Harvest cells, extract mRNA, hybridise onto sequences of 1800 genes, sequence and visualise expression.

Green= high expression 
Red= low expression

Question 2

How do you use the abundance of proteins to study differential gene expression?

Answer 2

Isolate specific cell types, lyse cells to release proteins, separate proteins on a 2D gel and stain the separated proteins and visualise.

Blue dots are unique protein
Red dots are common protein

Question 3

How can you study differential gene expression?

Answer 3

By measuring the abundance of mRNAs, by looking a protein abundances.

Question 4

What are the four major properties of cells?

Answer 4

Differentiation, specialisation, interaction and movement.

Question 5

How is different expression of genes possibly caused?

Answer 5

Gene loss, gene amplification, same genes expressed at different levels.

Question 6

What experiments proved that genes were not lost during development?

Answer 6

Differentiated cells in fogs, carrot and cows still contains all genes required to build a complete organism.

Question 7

What are changes in gene expression due to gene amplification?

Answer 7

Very rare, eg. Chorion genes in drosophila fruit flies and xenopus eggs ribosomal RNA genes

Question 8

What is differential gene expression cause mostly by?

Answer 8

Changes in in transcription of an unchanging set of genes.

Question 9

How is transcription controlled?

Answer 9

Gene regulatory sequences and binding proteins; all genes have control regions that recruit RNA polymerase and regulator proteins whose binding determines the level of transcription.

Question 10

What varies between different cell in an organism?

Answer 10

Their regulatory proteins, not their genes.

Question 11

How can you detect transcripts via hybridisation?

Answer 11

Northern blot analysis, in situ hybridisation, quantitative PCR and microarrays.

Question 12

Give the brief steps and benefits of northern blot analysis?

Answer 12

RNAs separated by PAGE and transferred to filter and radio-labelled probes hybridises to RNA and the probe is detected by autoradiography.
Very direct method and it can be quantitative.

Question 13

Briefly describe the steps and benefits of in situ hybridisation?

Answer 13

Tissue is prepared by fixing and permeabilisation, a labelled DNA or RNA probe and detect by autoradiography or microscopy.
Benefit: can show location of transcript expression in whole tissues and intact organisms

Question 14

Briefly describe the steps and benefits of quantitative PCR?

Answer 14

First step is reverse transcription of mRNA or cDNA, then PCR with fluorescent labelled primers or dye that binds dsDNA. Fluorescence is emitted when a dsDNA PCR products is made and the rate of appearance relates to concentration of mRNA.
Benefits: quantitative rapid and can detect several targets in one tube (multiplex).

Question 15

Briefly describe the steps and benefits of microarrays?

Answer 15

DNA oligos representing 1000s of genes immobilised on chip, cell mRNAs copied to cDNA, labeled with red or green dye, and the cDNAs are hybridised washed and scanned
Benefits: can detect many hundred transcripts simultaneously, highly specific bidding can be achieved- SNP analysis, can detect genetic material in samples.

Question 16

How can you detect protein expression?

Answer 16

Using a reporter, an easily detectable protein which can be detected when the gene is expressed.

Question 17

What are the two types of reporter genes?

Answer 17

Fluorescent for visualising live cells or enzyme for high sensitivity.

Question 18

What does the choice of reporter depend on?

Answer 18

Largely on use and whether the organism already expressed the reporters.

Question 19

Describe GFP?

Answer 19

Natural product of aequorea victoria jelly fish, 238 amino acids long, 11 strand beta barrel, central chromophore, no required cofactors or substrate, used in living cells but has a 1 hour time lag due to folding.

Question 20

Give some other common reporters?

Answer 20

ß-galactosidase- changes blue with X-Gal substrate
ß-glucoronidase- changes blue with X-Gluc substrate
Luciferase- needs a lucifer in substrate.

Question 21

What is the even skipped gene (Eve)?

Answer 21

Essential gene in drosophila development to define body segment formation acting very early on in development. Well characterised regulation and expression

Question 22

What controls Eve expression?

Answer 22

A long upstream region of 7.3 kb which controls where and when.

Question 23

What are two important research questions when looking at Eve?

Answer 23

What sequences are important for expression of each stripe and what regulatory protein bind to these sequences

Question 24

How can you define the sequence in four steps?

Answer 24

1) substitute Eve orf for a reporter orf to see where the gene is expressed
2) make deletions in the 7.3 kb regulatory region
3) introduce these altered genes into drosophila eggs
4) ask, does genes expression still occur in stripe 2 in developing embryos?

Question 25

How are the deletions made in step 2) of defining the sequence or Eve made?

Answer 25

Restriction enzymes, nuclease digestion or site directed mutagenesis.

Question 26

How would restriction enzymes introduce a deletion?

Answer 26

Map the 7.3 kb region and make deletions in regions until a deletion is made which is in a region that shows a visible result.

Question 27

How would nuclease digestion insert substitutions?

Answer 27

Exonuclease 3 digests 3’ termini of an exposed duplex DNA, therefore make deletions until a deletion is made which is in a region that shows a visible result.
Bal 31 digests both strands of an exposed duplex DNA.

Question 28

How would site directed mutagenesis introduce a deletion?

Answer 28

Can make single nucleotide changes to precisely define required sequences.
Design complimentary oligos with mismatches at desired position, extend oligos in PCR and digest wild type with Dpn1, transform mutant PCR products into bacteria and check for it by sequencing.

Question 29

What four main methods are used to study regulatory proteins?

Answer 29

Gel mobility shift analysis and affinity chromatography for what proteins and DNA footprint analysis and chromatin immunoprecipitation for where they bind.

Question 30

What does gel mobility shift analysis depend on?

Answer 30

DNA moving in proportion to its size on a gel, that the movement of DNA is slower when bound by protein and that DNA binding proteins are highly specific for DNA sequences.

Question 31

Give the steps of a gel mobility shift assay?

Answer 31

1) make a hot DNA fragment of the regulatory regions
2) fractionate cell proteins by centrifugation
3) mix fractions with hot DNA fragment
4) allow the regulatory proteins bind the hot DNA
5) run on a gel
A shift in mobility indicates that a cell has bound to the hot DNA regulatory protein

Question 32

How can you find out what the protein is when you have performed a gel mobility shift assay?

Answer 32

Can use an antibody to find out what the protein is = super shift! or use mass spectrometry analysis.

Question 33

What does DNA affinity chromatography depend on?

Answer 33

DNA binding proteins being highly specific for DNA sequences.

Question 34

Give the steps of DNA affinity chromatography?

Answer 34

1) make DNA fragment of the regulatory region
2) attach to a solid matrix, eg. Agarose
3) add cell lysate to column and allow for the regulatory proteins to bind to the immobilised DNA
4) elute with salt
5) analyse with mass spectrometry
Can identify which proteins bind but not where.

Question 35

Give an example of how DNA footprint analysis is carried out for hunchback regulatory protein?

Answer 35

1) Purify hunchback
2) Make hot regulatory region
3) Bind DNA and hunchback
4) Add DNase 1 which cuts once
5) Hunchback will protect the binding site
6) Run hot DNA fragments on gel
7) Identify regions with hunchback binding sites.

Question 36

Give an example of how chromatin immunoprecipitation is carried out for hunchback regulatory protein?

Answer 36

1) Treat living cells with formaldehyde or UV to cross link
2) Extract genomic DNA-protein complex
3) Cleave DNA into 300 bp fragments using sonification
4) Purify with antibodies
5) Remove the protein from the DNA-protein complexes
6) Determine the nucleotide sequence
7) Deduce binding site

Question 37

What are the regulatory proteins involved in Eve expression?

Answer 37

Hunchback, bicoid, kruppel and giant

Question 38

For expression of Eve in stripe 2 at what levels do the regulatory proteins have to be?

Answer 38

Bicoid and hunchback have to be high and Kruppel and Giant have to be low.

Question 39

What do the gene regulatory regions also specify gene expression levels with respect to?

Answer 39

Time

Question 40

In multicellular regions what do gene regulatory regions and their biding proteins control in gene expression?

Answer 40

Their level, location and time

Question 41

What methods are their to get a reporter construct to express in mammals?

Answer 41

Pro-nuclear micro injection and embryonic stem cell transfection

Question 42

How do you use pro-nuclear micro injection to express a reporter gene construct in mammals?

Answer 42

Inject copied of the gene into a fertilised egg before the fusion of a nuclei at the single cell stage and insert the embryo into a surrogate mother and the offspring will hopefully carry the transgene.

Question 43

When can you screen for the transgene germ line after using pro-nuclear microinjection?

Answer 43

After the second generation as after the first generation only expression is shown in the transgene.

Question 44

What are the disadvantages of using pro-nuclear micro injection to insert a transgene?

Answer 44

Time consuming, have to wait for second generation and sexual maturity. Costly. Success rate is low as site of insertion is random.

Question 45

How do you use embryonic stem cell transfection to express a reporter gene construct in mammals?

Answer 45

Transgene introduced to the stem cells in culture, cells from colonies that can be screened, combine the embryonic stem cells with transgene and early embryo forming a hybrid embryo. The hybrid is incubated in a female mouse and the offspring are screened for expression and second generation is screened for germ-line expression

Question 46

What are the major advantages with embryonic stem cell transfection?

Answer 46

No need for initial micro infection and screening does not require a live animal.

Question 47

How are genes transferred in plants?

Answer 47

Using a bacteria- agrobacterium tumefaciens, which naturally infects plants and transfers it’s DNA mediated by a plasmid Ti and depends on T-DNA repeats.

Question 48

What is the main aim of generating a transgenic organism?

Answer 48

To study gene function during development.

Question 49

How can you get plasmids into cells?

Answer 49

Transfection with liposomes or calcium phosphate,
Electroporation
Bombardment

Question 50

How is transfection with liposomes performed?

Answer 50

Liposomes are made from cationic detergents, spherical lipid bilayer with an aqueous interiors which fuse with plasma membrane and have very high transfection efficiency.
They are commercially available but expensive.

Question 51

How is transfection using calcium phosphate performed?

Answer 51

Only works in certain cell types and has low efficiency, 30-50%. DNA precipitates and binds to the membrane and is taken up by an unknown mechanism. It is relatively inexpensive.

Question 52

How is Electroporation performed?

Answer 52

Cells are placed in an electrical field and pores are formed on the plasma membrane which allows for the DNA to diffuse into the cell.
Has high efficiency and can be used of reactive animal cells, or treated plant or yeast cells.

Question 53

How is bombardment performed?

Answer 53

Fires 0.3-3.6 um gold particles coated with DNA or RNA and can be used for delivery into live organism.

Question 54

What is a method of studying gene or protein function when they are not required for development?

Answer 54

To prevent its correct expression using knock out or knock in mutants.

Question 55

What is the basis of knock out mutants?

Answer 55

To replace the the functional gene with one that is non-functional using homologous recombination. The gene can be inactivated by deletion if orf, insertion of stop codons or deletion of start codons.

Question 56

What are the advantages of knock in/out mice?

Answer 56

An study whole organisms, little ambiguity in results but cost and time can be an issue.