Molecular Genetic Techniques 3 (L7) Flashcards
why study proteins in bacteria as opposed to animals?
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
Lac operon
cluster of genes to help bacteria metabolize lactose - normally has a repressor bound to the operator (non-fxnal)
what is the preferred energy source for bacteria?
glucose
what happens with lac operon w/ low glucose?
allolactose binds repressor -> not bound on operator -> now RNA pol can activate all genes in Lac operon to help metabolize lactose
IPTG
analog of allolactose
clinical application of lac operon
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)
why use eukaryotic cells instead of viral for protein expression?
to get stable transformation
transient transfection
expression decreases over time b/c viral origin of replication is not always faithful, so over time, don’t get as much expressed
stable transfection (transformation)
vector integrated into host genome to get faithful replication
transient vs. stable transfection: where is protein expressed from
transient: from cDNA in plasmid DNA
stable: from cDNA integrated into host chromosome
structural analysis of proteins
X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy
lysosomal storage diseases
disorders that result from abnormal metabolism of substances such as glycosphingolipids, glycogen, mucopolysaccharides, and glycoproteins
Gaucher disease
glucocerebrosidase deficiency - prevalent in Ashkenazi jews
diagnosis of Gaucher’s disease
enzyme assay: if enzyme activity in patient drops below 30% of normal level -> problem -> genetic testing using automated fluorescence dideoxy sequencing methods
forward genetics
means of identifying a genotype that is responsible for a phenotype (can consider the disease gene identification forward genetics)
mutagenesis
mutants created in cells and animals using a toxin substance that would induce random mutation
yeast
temperature sensitive mutants (conditional mutants)
complementation analysis using double mutants
used to indicate whether mutations are on the same gene or different genes
suppression
mutation in both alleles leads to suppressed mutant
synthetic lethality
mutation in both alleles leads to severe defect or no product made
yeast-two-hybrid system
- Tag protein (X) in one plasmid with DNA-binding domain
- Tag protein (Y) in another plasmid with activation domain
- 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
does gene expression pattern tell you location of protein expression?
not necessarily - find out by promoter-fusion or protein-fusion
promoter fusion
when you translate the protein, GFP is attached to the promoter
protein fusion
link GFP gene right next to ODR sequence - ODR linked w/ GFP when expressed
what can spatial expression pattern tell you about a protein?
some information about the function of the protein
what is the cellular expression pattern needed for?
tells where the gene is expressing and where protein is found - important for disease-causing genes
reverse genetics
inactivate a gene so that you can study the phenotype to determine function
gene inactivation
- 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
- create a disruption construct
- transform construct into diploid cell
- cell will select for kanMX gene (replaces target gene)
If disrupted gene is essential, half of the spores will be nonviable
totipotent
cells from early embryo that can give rise to all embryonic and placental tissues
pluripotent
cells that form all embryonic tissues, but no placental tissues
origin of embryonic stem cells
inner cell mass after 64 cell stage
outcomes of positive and negative selection of recombinant ES cells
- homologous recombination - gene-targeted insertion -> mutation in gene X; cells resistant to G-418 and ganciclovir (thymidine kinase gene didn’t get into genome)
- 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)
formation of ES cells carrying a knockout mutation
- take cells (nonrecombinant, recombinants with random insertion, and recombinants with gene-targeted insertion)
- treat with neomycin (positive selection) -> kills nonrecombinants
- 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
what does chimeric mean?
containing cells from multiple embryos
generation of chimeric mice
- remove zona pellucida of embryos of two differently colored mice
- adhere blastocysts -> fuse to form a chimeric blastocyst
- chimeric blastocyst implanted into surrogate foster mother
- chimeric pup
generation of knockout mice
- Inject ES cells into blastocoel cavity of early embryos (brown A/A, X-/X+ and black a/a, X+/X+)
- Surgically transfer embryos into pseudopregnant female.
- Possible progeny: chimeric or those w/ no ES incorporated.
- 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+) - ES cell-derived progeny will be brown (half of them will have X- and the other half X+)
- Progeny from ES cell-derived germ cells are selected
- Screen brown progeny DNA to identify X-/X+ heterozygotes.
- Mate X-/X+ heterozygotes.
- Screen progeny DNA to identify X-/X- homozygotes -> knockout mice
knockout genotype
X-/X-
new genome-editing tools for medicine
ZFN - zinc finger nuclease
TALEN - TAL effector nuclease
CRISPR/CAS - CRISPR-associated nuclease
two basic mechanisms that can edit genome
NHEJ (results in chromosomal deletion or gene disruption)
Homology-directed repair (results in gene correction or targeted integration)
conditional knock out mice
KO in specific cell type at the right time
cre-lox recombination
loxP-cre mouse: all cells carry one copy of loxP-modified gene X, one copy of gene X knockout, and cre genes
gene (over)expression by transgenesis
- inject foreign DNA into one of pronuclei of fertilized mouse egg prior to fusion of male and female pronuclei
- transfer injected eggs into foster mother
- about 10-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line
- breed mice expressing foreign DNA to propagate DNA in germ line
application of (over)expression by transgenesis
over-expressing a protein or a dominant-negative protein;
expressing a reporter construct
