Molecular Genetics

Question 1

What important element of cell division is missing in oocytes?

Answer 1

centrosomes (centrioles), oocytes rely on motor proteins (dynein) to organize, centrioles are created from the basal bodies of the sperm flagella

Question 2

What is DNA transfection?

Answer 2

adding DNA to cells that are growing in a tissue culture environment

Question 3

tissue culture cells

Answer 3

cells grown in media in a culture dish that can divide indefinitely

Question 4

primary cells

Answer 4

cells from a normal animal temporarliy maintained in a culture dish

Question 5

transient transfection

Answer 5

introduce circular DNA plasmid into a cell and it is NOT incorporated into the genome, therefore it will not be replicated and passed on (2 day lifespan)

Question 6

stable transfection

Answer 6

introduce linear DNA into a cell with the goal of integrating the DNA into the genome tomake a uniform and permanent cell line. Requires a marker to ensure positive selection of transfected cells

Question 7

stable transfection mechanism

Answer 7

add linear DNA to cell in a high ratio (gene of interest to resistance gene, each has necessary promoters..etc)

Question 8

3 ways to insert plasmids into cells for transient transfection

Answer 8

DEAE dextran and Calcium phosphate (pinocytosis), liposomes, electroporation

Question 9

reasons for doing transient transfection

Answer 9

quick (2 days) and reproducible analyses of the regulatory elements on the plasmid or the gene product made from the plasmid

Question 10

reason for doing stable transfection

Answer 10

to establish a permanent cell line, but it is time consuming (30 days +)

Question 11

what is required to grow a plasmid in bacteria cells?

Answer 11

bacterial origin, drug resistance genes, and the gene of interest

Question 12

what is required to do stable transfection in eukaryotic cells?

Answer 12

regulatory elements (enhancers, promoter), gene of interest (cDNA), polyadenylation sites, separate drug resistance gene w/ enhancer/promoter and polyA sites

Question 13

What is the advantage to usign viral agents for transfection?

Answer 13

efficient!

Question 14

Why is it important to use eukaryotic cells to express recombinant DNA?

Answer 14

to produce a protein that has undergone posttranslation modifications

Question 15

Through what mechanism do genes intergrate into the genome in stable transfection?

Answer 15

non-homologous end joining repair pathway (inefficient 1/10,000-1,000,000 integrate), use antibiotic resistance to select for the integrated cells

Question 16

Positive Selection

Answer 16

cells that have a resistance gene will live in the presence of the drug, cells that do not have the gene will die

Question 17

How do you produce recombinant viral products?

Answer 17

you must infect the gene of interest viral vector with a plasmid encoding gag, pol and env (genes to create the retroviral structure w/eukaryotic enhance/promoter) into a cell to create the viral products

Question 18

How does the viral vector transfect other cells?

Answer 18

once made, the vector will have the gene of interest and necessary retroviral elements, but not the genes to make new viruses. So when the virus enters a new cell, RNA–> DNA and is incorporated into the genome and the infection will stop.

Question 19

What are some 4 limitations to retroviral gene delivery?

Answer 19

1. silencing of the retroviral promoter/enhance over time (LTR) 2. size limitations (t pass through nuclear pore) 4. can disrupt important genes depending on where it integrates into genome

Question 20

2 strategies for viral integration

Answer 20

1. use viruses that don’t integrate into genome - episomes (won’t disrupt cells) 2. integrate into genome into primary cells or cell lines

Question 21

What are the advantages to the mouse model?

Answer 21

1. look at interactions within whole system (not just one tissue cell line) 2. rapid generation time, many inbred strains (genetically homogeneous) 3. eary housing needs

Question 22

how do you generate transgenic mice?

Answer 22

1. create transgene in bacteria w/ promoters/enhancers, introns/exons, polyA sites) 2. inject linear, dsDNA into male pronucleus of fertilized mouse egg 3. re-implant egg in mother 4. screen progeny for number of DNA copies to see if expression of the transgene occurs and if it has disrupted any important genes through integration (NO SELECTION PROCESS)

Question 23

what mouse models can you make with transgenic mice?

Answer 23

1. express a new, normal protein in the mouse 2. express a mutant or disease-causing protein in the mouse in tissues where it is usually found 3. mis-express a normal or mutant protein at a new time and place in the mouse

Question 24

knock-out mouse

Answer 24

gene has been disrupted or has a loss-of function mutation

Question 25

how can you make a knock-out mouse?

Answer 25

1. insert stop codon 2. insert foreign gene within the targeted gene 3. delete the gene

Question 26

knock-in mouse

Answer 26

mouse with a mutant protein created by introducing a mutatation in gene without disrupting the other parts of the gene

Question 27

When would a knock-in mouse model be used?

Answer 27

to mark or tag a protein and then follow its activity in a disease process, or follow a mutant protein

Question 28

What must be included in the recombinant DNA that you will insert to make a knockout mouse?

Answer 28

1. homolgous region to gene you want to knock out 2. region of dissimilarity in middle 3. positive and negative selection factors

Question 29

What type of cells are used to create knockout mice?

Answer 29

insert linear DNA segment into embryonic stem (ES) cells

Question 30

What cell process is used to transfer the linear DNA segment into the genome of the knockout mice?

Answer 30

homologous recombination

Question 31

What is the purpose of negative and positive selection factors in the creation of knock out mice?

Answer 31

positive factor tells you whether or not the knockout construct was integrated (will be resistant to a drug), negative factor tells you if the construct combined in the correct location via homolgous recombination or in a random place via nonhomologous end joining (in later case, neg. factor will still be present that makes them sesitive to a certain drug, so will die when drug is added)

Question 32

at the end of the process, what parts of the knockout construct should have integrated into the knockout mouse?

Answer 32

the homologous end regions, the dissimilar middle part of the gene, the positive selection factor (oftern in the middle part of the gene)

Question 33

chimeric mice

Answer 33

progeny born from ES cells of white animals injected into blastocyst of black-coated animals

Question 34

agouti mouse

Answer 34

progeny of a chimeric mouse (confirms ES derived) and a black coated mouse w/ 50% chance of having mutation

Question 35

embryonic stem cell

Answer 35

pluripotent cell from human embryoes, can be maintained in tissue culture, must be matched to recipient, can have tumorigenic properties

Question 36

induced pluripotent cells

Answer 36

cells from almost any tissue in the body can be induced to a pluripotent state, can be made from the individual’s cells (no matching problem)

Question 37

What are the steps of correcting a gene and replacing tissue in an animal model?

Answer 37

1. take cells from individual 2. induce pluripotency factors 3. use iPs clone to knock-in vector for wildtype/normal gene 4. select clone with succesful gene replacement 5. induce differentiation of desired tissue stem cells 6. transplant into animal