1b Molecular Biology Exam 2

Gene Expression Tools

What are the general attributes of model organisms?

Relatively cheap and plentiful

Inexpensive to house

Straightforward to propagate

Short gestation periods that produce large numbers of offspring

Easy to manipulate in the lab

Some have fairly small and relatively uncomplicated genome

What is the scientific name for mouse?

Mus musculus

Mouse have ______ of strains that are useful for studying

hundreds

What is often the first step of transfection?

Introduce genes or gene constructs into a cell line

In bacteria how are genes/gene constructs introduced into a cell line?

Transformation or Transduction

How is transformation mediated?

chemically or by electroporation

How is transduction mediated?

bacteriophage

What are the multiple methods by how eukaryotes undergo transfection?

Viral

Chemical

Physical

What is a recombinant virus?

gene cloned into viral genome

What is the order by how viral transfection occurs?

1. Gene cloned into viral genome

2. Viral particles are amplified using “packaging cell line”

3. Viral particles are purified and titered

4. Viral particles are allowed to infect target cells

Chemical transfection is not a chemical _______

reactions

How does DNA enter the cells in chemical transfection

via carrier molecules

Nucleic acids are _____ charged

negatively

Carrier molecules are ______ charged

positively

What are two types of carrier molecules?

Polymers

Lipids

How are nucleic acids taken up via chemical transfection?

endocytosis

What are three types of chemical transfection?

Diethylaminoethyl-dextran (DEAE)-dextran

Calcium phosphate

Lipofection

Diethylaminoethyl-dextran (DEAE)-dextran:

Polycationic and binds to DNA

Cheap

Simple

Low efficiency

Some toxicity

Calcium Phosphate:

Mix DNA with CaCl2

Add to a saline-phosphate solution

DNA-calcium phosphate co-precipitates

Precipitate sticks to cells

Uptake by endocytosis

Cheap

Low efficiency

Some toxicity

Lipofection:

Most commonly used method

Cationic lipids used

Lipids absorb to cellular membranes

Higher efficiency

Relatively cheap

DNA/RNA size does not matter

What are the four types of physical transfection?

Electroporation

Microinjection

Microballistics

Biolistics

Electroporation:

Frequently used

Cells and DNA are suspended in a buffer

Pulsed with very high voltages

Membrane pores are induced

Works well for large constructs

High morality rates

Why does electroporation have high mortality rates?

Voltage can kill

Cells might not recover by fixing their membranes

Microinjection:

Use a micromanipulator and microscope

Works at a single-cell level

Nearly 100% efficiency

Low throughput

Requires skills operator

Expensive equipment

Microballistics:

Microparticles made with Au or W

DNA is coated onto microparticles

Voltage or gas pressure is used to propel particles (gene gun)

Effective with plants with walls intact

Used with whole plant/animal tissue

Works well for agricultural applications (vaccinations)

No known limit on size of genes or number of genes

Very high mortality

Need lots of starting cells

Transient transfection:

Make construct without selectable marker

Transfect cells

Incubate to allow expression

Assay gene expresion

Stable Transfection is good for ____ term

long

Stable transfection:

Make construct with selectable markers

Transfect cells

Select for recombinant cells

Analyze for gene expression

Reporter genes can

visualize what is going on in the cell

Reporter Genes:

Known gene whose RNA or protein concentration can be measured easily and accurately

Reporter genes can replace other ________ whose products are difficult to measure quantitatively

coding regions

What are the applications of reporter genes?

Activity of regulatory regions

Intracellular fate of a gene product

Protein-protein interactions

DNA-protein interactions

Promoter bashing:

Reporter genes placed under control of suspected promoter

Mutations can be made, conditions can be changed

Then measure reporter gene expression levels

What are some examples of reporter genes with bacterial origins?

Beta-galactosidase (lacZ)

Beta-glucoronidase (gus)

Chloramphenicol acetyltransferase (cat)

Beta-glactosidase can be measured with:

ONPG (yellow product)

X-gal (blue product)

What color is the product of Beta-glucoronidase

blue

Chloramphenicol acetyltransferase is good in vitro with ____-

TLC

Generally speaking, the more intense the band the

more gene expression

What are some examples of reporter genes with eukaryotic origins?

Luciferase (luc)

Green fluorescent protein (gfp)

Where does Luciferase (luc) come from?

Photinus pyralis

Luciferase (luc) of Photinus pyralis:

Oxidizes luciferin

Generates bioluminescence

Where the the green fluorescent protein (gfp) come from?

Aequorea victoria

Green fluorescent protein (gfp) from Aequorea victoria:

Autofluorescent

Can be used for living cells

Reporter genes can be _____ (____) with protein to be studied

fused (in frame)

What happens when a reporter gene is fused with the protein to be studied?

It allows the reporter to follow the gene product which can help determine the location of product and expression

Recombinant proteins can be ______

overexpressed

How are recombinant proteins overexpressed?

The gene is cloned into an expression vector which induces for expression. The vector has a promoter that allows the cloned gene to be expressed

When is relates to the over expression of recombinant proteins, what might the vector have?

Reporter fusion and/or purification tag

What are some examples of bacterial vectors:

lac promoters

pBAD vectors

pET vectors

lac promoters:

works exactly like the induction of lac

Add IPTG, and cells begin to express protein

pBAD vectors:

promoter from araBAD operon

Add arabinose

pET vectors:

Promoter from phase T7 RNA pol fused to lac

Add IPT, and T7 RNA pol will express gene

Proteins are often ______ so they can be purified

tagged

How are proteins tagged?

1. Gene is fused to purification tag in specialized vector

2. Grow cells and induce expression (over expression)

3. Gently lyse cells

4. Run lysate through a resin column that selectively binds to tag

5. Wash away proteins attached due to non-specific binding with a buffer

6. Elute fusion protein off of column (differs by the tag)

The 5’ or 3’-end of protein has ____ histidine residues

six

What type of columns are his tags run through?

Nickel column

What is the procedure for his tags?

1. Run through a resin column with nickel

2. Most non-tagged proteins should pass through

3. Imidazole then used to elute protein from column hat W

What is another name for GST fusions?

GST Pulldown

What does GST stand for?

glutathione-S-transferase

Where is GST fusion added?

to the 5’-end of protein-coding gene

In GST fusions, what column is used

glutathione column

What is the substrate of GST?

glutathione

What are the two ways the GST fusion can be eluted?

Excess glutathione can be added to complete with resin-bound glutathione

Sequence specific protease can be added to digest GST fusion, then the over-expressed protein will be released from the column

For immunotags, specialized vectors can contain _____ tags at 5’- or 3’-end

epitope

What type of columns are used for immunotags?

resin columns with Protein A from Staph

What are the steps of immunotags?

1. Run through a resin column with Protein A from Staph

2. Antibodies are bound non-specifically

3. Antibodies specific for tag are attached to Protein A resin

4. Lysate is passed over resin

5. Antibodies grab immunotag

6. Then elute with excess antigen

Genes can be ____ or _____

Specific

Random

Three main types of in vitro mutagenesis/

Deletion mutagenesis

Linker-scanning mutagenesis

Site-directed mutagenesis

Deletion mutagenesis uses PCR to:

Amplify part of a suspected promoter element, or

Amplify all of the suspected promoter element, then digest with a restriction enzyme

In deletion mutagenesis you _____ the remaining fragments of the promoter into a vector

Subclone

In deletion mutagenesis, how do you tell which fragments of the promoter influence expression, under certain conditions, with certain TFs?

By attaching to a reporter gene

Site-directed mutagenesis is nearly

random

In Site-directed mutagenesis, you can use _____ to introduce specific point mutations, multiple mutations, or indels

PCR

In site-directed mutagenesis, mutations are only made ______ the primers, not outside of them

within

Mutations bind _____ and _____ of the gene of interest on a primer

upstream

downstream