Molecular Genetic Techniques 3 (L7)

Question 1

why study proteins in bacteria as opposed to animals?

Answer 1

in animals, you have post-translational modifications that do not happen in bacteria - need to express protein in form that will be functional in vivo

Question 2

Lac operon

Answer 2

cluster of genes to help bacteria metabolize lactose - normally has a repressor bound to the operator (non-fxnal)

Question 3

what is the preferred energy source for bacteria?

Answer 3

glucose

Question 4

what happens with lac operon w/ low glucose?

Answer 4

allolactose binds repressor -> not bound on operator -> now RNA pol can activate all genes in Lac operon to help metabolize lactose

Question 5

IPTG

Answer 5

analog of allolactose

Question 6

clinical application of lac operon

Answer 6

clone a target gene -> transform into plasmid in the lac operon -> add IPTG to induce lac operon activation -> increase protein translation of the target protein -> isolate protein from bacteria (used to make many of the proteins used in clinic)

Question 7

why use eukaryotic cells instead of viral for protein expression?

Answer 7

to get stable transformation

Question 8

transient transfection

Answer 8

expression decreases over time b/c viral origin of replication is not always faithful, so over time, don’t get as much expressed

Question 9

stable transfection (transformation)

Answer 9

vector integrated into host genome to get faithful replication

Question 10

transient vs. stable transfection: where is protein expressed from

Answer 10

transient: from cDNA in plasmid DNA
stable: from cDNA integrated into host chromosome

Question 11

structural analysis of proteins

Answer 11

X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy

Question 12

lysosomal storage diseases

Answer 12

disorders that result from abnormal metabolism of substances such as glycosphingolipids, glycogen, mucopolysaccharides, and glycoproteins

Question 13

Gaucher disease

Answer 13

glucocerebrosidase deficiency - prevalent in Ashkenazi jews

Question 14

diagnosis of Gaucher’s disease

Answer 14

enzyme assay: if enzyme activity in patient drops below 30% of normal level -> problem -> genetic testing using automated fluorescence dideoxy sequencing methods

Question 15

forward genetics

Answer 15

means of identifying a genotype that is responsible for a phenotype (can consider the disease gene identification forward genetics)

Question 16

mutagenesis

Answer 16

mutants created in cells and animals using a toxin substance that would induce random mutation

Question 17

yeast

Answer 17

temperature sensitive mutants (conditional mutants)

Question 18

complementation analysis using double mutants

Answer 18

used to indicate whether mutations are on the same gene or different genes

Question 19

suppression

Answer 19

mutation in both alleles leads to suppressed mutant

Question 20

synthetic lethality

Answer 20

mutation in both alleles leads to severe defect or no product made

Question 21

yeast-two-hybrid system

Answer 21

1. Tag protein (X) in one plasmid with DNA-binding domain
2. Tag protein (Y) in another plasmid with activation domain
3. Put into cell.
   If X and Y bind to each other in cell, brings bound proteins together - DBD will recognize DNA promoter, then AD will activate transription (Bait = X and prey = Y) - if many different Y’s interact with X, expand knowledge of target gene and how it interacts with other proteins inside the cell

Question 22

does gene expression pattern tell you location of protein expression?

Answer 22

not necessarily - find out by promoter-fusion or protein-fusion

Question 23

promoter fusion

Answer 23

when you translate the protein, GFP is attached to the promoter

Question 24

protein fusion

Answer 24

link GFP gene right next to ODR sequence - ODR linked w/ GFP when expressed

Question 25

what can spatial expression pattern tell you about a protein?

Answer 25

some information about the function of the protein

Question 26

what is the cellular expression pattern needed for?

Answer 26

tells where the gene is expressing and where protein is found - important for disease-causing genes

Question 27

reverse genetics

Answer 27

inactivate a gene so that you can study the phenotype to determine function

Question 28

gene inactivation

Answer 28

1. homologous recombination in yeast to design a set of primers that are hybrid: 3’ end always complementary w/ antibiotic-resistant gene; 5’ end always complementary to target gene
2. create a disruption construct
3. transform construct into diploid cell
4. cell will select for kanMX gene (replaces target gene)
   If disrupted gene is essential, half of the spores will be nonviable

Question 29

totipotent

Answer 29

cells from early embryo that can give rise to all embryonic and placental tissues

Question 30

pluripotent

Answer 30

cells that form all embryonic tissues, but no placental tissues

Question 31

origin of embryonic stem cells

Answer 31

inner cell mass after 64 cell stage

Question 32

outcomes of positive and negative selection of recombinant ES cells

Answer 32

1. homologous recombination - gene-targeted insertion -> mutation in gene X; cells resistant to G-418 and ganciclovir (thymidine kinase gene didn’t get into genome)
2. nonhomologous recombination - random insertion -> no mutation in gene X; cells resistant to G-418 but sensitive to ganciclovir (thymidine kinase present, but doesn’t replace gene X - so get neomycin resistance but also get tk)

Question 33

formation of ES cells carrying a knockout mutation

Answer 33

1. take cells (nonrecombinant, recombinants with random insertion, and recombinants with gene-targeted insertion)
2. treat with neomycin (positive selection) -> kills nonrecombinants
3. treat with ganciclovir (negative selection) -> kills recombinants that got tk from random insertion
   End up with cells that have homologous recombination w/ gene of target knocked out

Question 34

what does chimeric mean?

Answer 34

containing cells from multiple embryos

Question 35

generation of chimeric mice

Answer 35

1. remove zona pellucida of embryos of two differently colored mice
2. adhere blastocysts -> fuse to form a chimeric blastocyst
3. chimeric blastocyst implanted into surrogate foster mother
4. chimeric pup

Question 36

generation of knockout mice

Answer 36

1. Inject ES cells into blastocoel cavity of early embryos (brown A/A, X-/X+ and black a/a, X+/X+)
2. Surgically transfer embryos into pseudopregnant female.
3. Possible progeny: chimeric or those w/ no ES incorporated.
4. Select chimeric mice for crosses to wild-type black mice
   (possible germ cells from brown: A/X+, A/X-, a/X+; from black: a/X+)
5. ES cell-derived progeny will be brown (half of them will have X- and the other half X+)
6. Progeny from ES cell-derived germ cells are selected
7. Screen brown progeny DNA to identify X-/X+ heterozygotes.
8. Mate X-/X+ heterozygotes.
9. Screen progeny DNA to identify X-/X- homozygotes -> knockout mice

Question 37

knockout genotype

Answer 37

X-/X-

Question 38

new genome-editing tools for medicine

Answer 38

ZFN - zinc finger nuclease
TALEN - TAL effector nuclease
CRISPR/CAS - CRISPR-associated nuclease

Question 39

two basic mechanisms that can edit genome

Answer 39

NHEJ (results in chromosomal deletion or gene disruption)

Homology-directed repair (results in gene correction or targeted integration)

Question 40

conditional knock out mice

Answer 40

KO in specific cell type at the right time

Question 41

cre-lox recombination

Answer 41

loxP-cre mouse: all cells carry one copy of loxP-modified gene X, one copy of gene X knockout, and cre genes

Question 42

gene (over)expression by transgenesis

Answer 42

1. inject foreign DNA into one of pronuclei of fertilized mouse egg prior to fusion of male and female pronuclei
2. transfer injected eggs into foster mother
3. about 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line
4. breed mice expressing foreign DNA to propagate DNA in germ line

Question 43

application of (over)expression by transgenesis

Answer 43

over-expressing a protein or a dominant-negative protein;

expressing a reporter construct