Lesson 17: Application of Molecular Biology

Question 1

What is a reporter gene?

Answer 1

Something that can be easily visualized in a cell.

Common reporter genes are:
- Jellyfish : Green Fluorescent Protein (GFP)
- emits fluorescent light when applied
- E.coli: Lac Z (beta-galactosidase)
- turns compound blue
- FIrefly: Luciderase Enzyme
- light producing chemical reaction

Question 2

What is the function of a reporter gene?

Answer 2

1) Show which cells express a gene

2) Used to determine regulatory sequences

Question 3

How are reporter genes constructed?

Answer 3

A coding sequence for a gene you want to express is REPLACED with a reporter gene.

• NOTE: The pattern of expression of the reporter gene should match the pattern of expression of the original coding sequence

Question 4

How are fusion proteins constructed?

Answer 4

Fusion proteins attach to the particular protein you are trying to localize in a cell. This type of experiment is done in a LIVING cell/organism. So you can treat with a drug or put the cell under different environmental conditions.
- NOTE: When you “fuse” two proteins together in the genome, we HAVE to consider the reading frame.

Question 5

What is the function of a fusion protein?

Answer 5

Allows you to see where a particular PROTEIN is LOCALIZED in a cell. This allows you to learn something about the protein and what its function might be.

Question 6

What is a fusion protein?

Answer 6

A fusion protein is a protein that is naturally fluorescent.
Ex: GFP (shine blue light and emit green light)
- When you mutate GFP other fusion proteins can be discovered (change color of light that is emitted).

Question 7

What are protein domains?

Answer 7

Region of a polypeptide chain that can usually fold independently and typically has its own cellular function or creates structure for the protein.

Question 8

What is an advantage of protein domains?

Answer 8

Through evolution, scientists learned that you can MIX and MATCH these protein domains.
- ex: as few as 1000 distinct exons of 30-50 amino acids could be mixed and matched to create hybrid genes in the human genome.

Question 9

What is exon shuffling?

Answer 9

RARE EVENT! (happens on evolutionary scale)
- Exons can swap/exchange in intron regions and will get spliced into the existing exons.
- As long as the splicing doesn’t alter the reading frame the new exon will become part of the protein and because introns are large and non-coding where the swap occurs is not critical.
—-> This allows a “mix and match” of protein domains and can create new proteins with new functions! Including transcription regulators. (evolutionary advantages)

Question 10

How does exon shuffling differ from alternative splicing?

Answer 10

Exon shuffling is much rarer, happens more frequently.

Question 11

What is the function of in situ hybridization?

Answer 11

Takes advantage of complementary base pairing to determine WHERE a gene is expressed in a cell or tissue.

Question 12

How does in situ hybridization work?

Answer 12

It uses a probe marked with a fluorescent or dye and then hybridization occurs in the tissue sample

Question 13

What are the advantages of in situ hybridization?

Answer 13

• Examine localization of cells expressing a particular mRNA

Question 14

What are the disadvantages of in situ hybridization?

Answer 14

• Must have a “fixed” sample (can’t be done in living organisms)
  
  • This differs from fusion proteins which are typically
    done in living organisms
• Need to know the mRNA sequence
• Limited to only a few mRNAs at a time

Question 15

When do we use in situ hybridization?

Answer 15

When we want to see where a PARTICULAR gene is expressed in an organism

Question 16

What is the function of RNA Seq?

Answer 16

Used to determine which genes are expressed and relative levels

Question 17

How does RNA Seq work?

Answer 17

1) Isolate RNA from samples
2) Fragment RNA into short segments
3) Convert RNA fragments into cDNA (DNA without introns)
4) Perform sequencing on cDNA
5) Map sequencing reads to the genome to determine which RNAs were in your sample and their relative levels.
- This also allows determination of alternative
splicing/isoforms

Question 18

What are the advantages of RNA Seq?

Answer 18

• Don’t need to know the sequence before
• Learn about what isoforms are expressed in a cell

Question 19

What are the disadvantages of RNA Seq?

Answer 19

• Expensive (but getting cheaper)
• Get a lot of data out of RNA Seq (sometimes its too much)

Question 20

When do we use RNA Seq?

Answer 20

When we want to know ALL the genes expressed in the cells

Question 21

What is the function of qPCR (quantitative, reverse transcriptase PCR)?

Answer 21

To quantify or determine the relative mRNA concentration

Question 22

How does qPCR work?

Answer 22

1) Make cDNA (template for PCR reaction)
- Start off with RNA. Then add a 5’ poly T primer which complementary base pairs with the poly A tail. Then reverse transcriptase will extend the primer and build the cDNA.

2) Denature (use heat to split the cDNA)

3) Design proper primers that can complementary base pair with DNA (primer annealing)

4) DNA polymerase extends from the primer, transcribing the DNA.
- Here is where dyes are used to quantify the amount of product that is being made

Question 23

What are the advantages of qPCR?

Answer 23

• Fast, relatively cheap
• Can be used to determine relative mRNA concentration (does it go up or down)
  
  • low PCR products -> low mRNA
  • high PCR products -> high mRNA

Question 24

What are the disadvantages of qPCR?

Answer 24

• Need to know the mRNA sequence
• Limited to only a few mRNAs at a time

Question 25

When do we use qPCR?

Answer 25

When we want to know if a SPECIFIC gene is expressed in cells

Question 26

What are methods for “breaking a gene”?

Answer 26

1) Knock down the mRNA level of the gene
- RNAi (siRNA)
2) Completely remove it or render it non-functional
- Gene knock-out (Homologous Recombination)
- CRISPR technology (CRISPR + NHEJ)
3) Generate mutations
- Clone and express mutants in a plasmid (Restriction
enzymes and PCR)
- CRISPR technology (CRISPR + HDR)

Question 27

How does RNAi technology knock down the mRNA level of a gene?

Answer 27

• Instead of using “foreign” RNA, we are going to introduce dsRNA of our choosing.
• This dsRNA will contain sequences against our gene of interest and will KNOCK DOWN the amount of mRNA and therefore protein being made by the cell.

Question 28

What are advantages of RNAi?

Answer 28

• Relatively cheap
• Easy to give to cells
• Does not require manipulation of the genome (only mRNA)

Question 29

What are disadvantages of RNAi?

Answer 29

• Not permanent (short-lived)
• Not a complete knock-out (may have residual expression) -> can confound results

Question 30

When do we use RNAi technology?

Answer 30

1) When we want to eliminate the mRNA/protein to learn about its function
2) When we want to only eliminate the mRNA/protein NOT THE GENE in the organism’s genome

Question 31

How do we make the dsRNA that we need for RNAi?

Answer 31

We can feed worms bacteria expressing dsRNA then they will release dsRNA.

Question 32

What is the function of gene knockout?

Answer 32

• At the DNA level, the DNA is changed to eliminate the entire or part of the DNA in the genome to render the gene non-functional
• Replace the gene with a selectable marker (which has a drug resistance gene). Only cells with the replacement will survive drug treatment. Thus, just keeping cells where we’ve made the knockout.

Question 33

How can we use homologous recombination for gene knockout?

Answer 33

1) Start off with an accidental double-strand break

2) Cell recognizes this break needs to be repaired.

3) Then we supply the cell with an identical donor DNA that has the selectable marker inserted in it
- We can trick the cell to repair the DNA using the homologous DNA with the selectable marker. So now the selectable marker will replace where the gene was.

Question 34

Describe general strategy for gene targeting in mice.

Answer 34

1) Culture embryonic stem cells from a mouse
2) Generate a targeting construct with regions of homology to to the target gene
3) Transfect targeting construct into embryonic stem cells
4) Select embryonic stem cells for their resistance to G418 and Ganciclovir
5) Grow population of embryonic stem cells carrying knock-out
6) Inject embryonic stem cells into a mouse blastocyst
7) Introduce into a pseudopregnant mouse
8) Isolate chimeric offspring
9) Mating between chimeric mouse and normal mice until you have mice only with the targeted gene

Question 35

What is the purpose of eukaryotic targeting?

Answer 35

It permits selection of cells that have properly generated the knock-out

Question 36

How do we use eukaryotic targeting for gene knock out?

Answer 36

When we do homologous recombination, we do not want any outside regions to be included, only the selectable marker (NeoR)

However, sometimes a random insertion (tkHSV) can occur while doing the gene knock out which is problematic.

Luckily, tkHSV is sensitive to ganciclovir, so these cells will die in the presence of ganciclovir. And the cells without tkHSV will survive since they are resistant to ganciclovir.

Question 37

What is NeoR?

Answer 37

makes the cells expressing it resistant to G418 (selectable marker)

Question 38

What is tkHSV?

Answer 38

(random insertion)
cells expressing this convert the compound ganciclover to a toxic compound – kills cells expressing it in the presence of ganciclover.
- Cells not expressing tkHSV are resistant to ganciclover

Question 39

What is CRISPR?

Answer 39

Clusters of Regularly Interspaced Short Palindromic Repeats

• This system functions in bacteria and acts like an “immune” system to target and destroy invading viruses.

Question 40

What is the function of CRISPR?

Answer 40

1) Generate gene “knock-out” mutations to study the function of a gene -> NHEJ
2) For precisely and permanently altering the genome of an organism -> HDR

• VERY EFFICIENT! Works in every cell type that its been tested in! Even cells that homologous recombination never work well in.
• With CRISPR we have the power to alter rapidly, cheaply, and easily any genome (even humans)

Question 41

How do CRISPR-Cas9 genetic scissors work?

Answer 41

Ca9 enzyme - endonuclease (“scissors”) - cuts DNA

1) Researchers artificially construct a single guide RNA (sgRNA) which forms a complex with the Cas9 and direct the scissors to the precise location in the genome where the cut will be made. (complementary base pairig is used also)

2) Cas9 will generate a double strand break in the DNA

Question 42

When do you want to use Non-Homologous End Joining (NHEJ)?

Answer 42

For a gene knockout
- NHEJ will insert or delete nucleotides (indels), thus causing gene disruption. So the gene’s function will be changed allowing for us to determine what that particular mRNA’s function was.

Question 43

When do you want to use Homology Directed Repair (HDR)?

Answer 43

For an insertion or repair of a mutation
- We want homology directed repair (HDR) to work to replace the gene with the mutant; supply a donor template