week 11 (recombinant dna tech) Flashcards
explain: human insulin production
pancreatic cells synthesize preproinsulin
⤷ precursor to insulin
⤷ doesn’t work until processed
- process:
⤷ cut to remove 24 N-terminal AA -> proinsulin
⤷ cut to remove another 35 AA
⤷ A and B chains join by disulfide bonds -> insulin
explain: two chain method of expression (expressing insulin in e.coli)
- synthetic genes for A and B chains were cloned and put into separate plasmids
- codons for methionine were put in between lacZ gene and each insulin gene
⤷ makes fusion gene
⤷ allows regulation over insulin by controlling lacZ - gene for insulin was put downstream to beta galactosidase
- cyanogen bromide cleaved C terminal of methionine
⤷ separates insulin in the fusion prot. - A and B chains are purified and mixed to make insulin
explain: transgene animals
- injecting vectors, mRNA, or prot. into embryos to insert foreign DNA
- need to be in right stage of dev. t survive the injection
- transgene needs to be incorporated into germline cells
⤷ transmissible to offspring - sounds easy but hard in practice
⤷ can kill cell
⤷ material might not enter
⤷ may not be incorporated into germline
explain: dolly the sheep
- example of cloning
- nucleus of an adult somatic cell injected into egg
- cell got shocked to stim. cell division
- not genetically identical to nucleus donor
⤷ very similar but has mitochondrial DNA from egg donor too
name: model organisms + what they’re used for (5)
E.COLI
- best for cellular lvl
- easy to maintain in lab
S.CEREVISIAE
- yeast
- good to grow and represents a euk. cell
C.ELEGANS
- simple multicellular life form
- clear (easy to observe)
D.MELANOGASTER
- fly
- good for genetics
- has neuronal circuits for studying complex beha.
M.MUSCULUS
- house mouse
- good mammal
- closest to humans
- more ethical boundaries
explain: forward genetics
- IDing genes by mutant phenotypes
- trial and error
- breeding indiv. and screening offspring for mutations
explain: F1, F2, F3 screening in forward genetics (exposed male to mutagen and bred w/ wt females)
- if mutation = dom., process is easier
- if mut. = dec, process is easier in org. that self-fertilize
F1 DOM
- dominant mutations appear in F1
- collect mutants and generate more pure breeding stock
F1 REC
- offspring will be carriers at best
- isolate and rebreed them to more wt indiv.
F2 REC
- collect and interbreed
F3 REC
- ID recessive mutants and collect to being pure breeding
explain: x-linked mutations in drosophila
- mut = attached x
- attached x are phenotypically female but their X chromo. are inherited together
- allows x-linked male mutants to pass x chromo to male offspring
if want to study an x linked trait:
- breed males w/ the trait to attached x females
- males pass the trait on X, females pass y
question: how to determine number of mutated genes in forward screen?
- crossing 2 recessive mut -> wt progeny = 2 separate genes mutated
- crossing 2 recessive mut -> mut progeny = 1 mutated gene
explain: reverse genetics
- begin w/ gene of interest and manipulate gene to ID mutant pheno.
- more common and easier now w/ new tech
- ex.:
⤷ CRISPR
⤷ gene knockout
explain: CRIPSR-Cas9 gene editing
- Cas9 = nuclease that makes double stranded breaks
- Cas9 needs guide RNA
⤷ complementary to genomic target
⤷ chooses where to cut - cut cells with either NHEJ or homologous recombination (SDSA)
- edit DNA by injecting embryo w/:
⤷ engineered guide RN
⤷ Cas9 mRNA
⤷ donor DNA template
explain: gene knockout
- deletion of the coding region of a gene
- done through CRISPR
- induces double stranded break at target gene
- supplies template w/ a marker
⤷ alters pheno. - marker allows confirmation of knockout
explain: gene knockout w/ RNA interference
- RNA i = used to silence a gene of interest
- insert gene that produces RNA that’s complementary to mRNA target
- if inserted gene is expressed -> gene will be silenced through mRNA degradation
explain: reporter genes
- indicates location of exp.
- gene can be reporter if it’s prot. product can be detected
- ex. GFP, lacZ
explain: enhancer trapping
- inserting a transgene w/ a weak promoter in a genome
- promotor is too weak to express the transgene
- if gene inserted near endogenous enhancer -> can express at high amounts
explain: GAL4-UAS system as an enhancer trap
- GAL4-UAS system = driver
- GAL4-UAS seq. = responder
- GAL4 = weak transcription factor
⤷ only regulates if it has UAS upstream - flies don’t have endogenous activators that bind to UAS or endogenous GAL4
⤷ need both transgenes
name: applications of GAL4-UAS
-
reporter system
⤷ monitor where gene of interest is expressed -
gene knockdown system
⤷ use GAL4 to express RNAi -
overexpression system
⤷ use GAL4 to express endogenous gene at higher amounts -
rescue system
⤷ use GAL4 to express gene in knockout
⤷ determine if wt pheno is rescued -
manipulate neurons
⤷ use GAL4 to express prot. that activate or silence neurons
explain: GAL4-UAS as a reporter system
- promotor fuse to GAL4
- UAS fuse to GFP (marker)
**promotor can be specific or general
⤷ ex. actin -> GFP everywhere
⤷ ex. specific neuron -> GFP only in specific neuron
- anywhere GFP is visible = shows where gene of interest is expressed
explain: GAL4-UAS and RNAi (knockdown)
- UAS fuse to RNAi construct
- promotor fuse to GAL4
- all cells w/ UAS and GAL4 get the RNAi construct -> prevents transcription
- ex. if promoter = actin, RNAi in all cells -> prevents prot. synthesis
- ex. inhibits pheromone genes in oenocytes
explain: GAL4-UAS for overexpression
- UAS fuse to gene you want to express
- GAL4 fuse to promotor (ex. actin)
- offspring w/ GAL4 and UAS will express the gene of interest
- using more restrictive promotor -> more regulated expression
- ex. upregulates pheromone genes in oenocytes (cells of insect)
explain: GAL4-UAS for forcing expression of a gene in knockouts (rescue)
- GAL4 fuse to promoter and gene deletion
- UAS fuse to gene and gene deletion
- offspring w/ GAL4 and UAS get deletion too
- but have GAL4 and UAS together -> forces expression of gene -> restores wt
explain: GAL80
- inhibitor of GAL4
- can be used as a driver to inhibit GAL4
⤷ instead of UAS
explain: GAL80 TS
- temp. sensitive GAL80
- enables temporal activation of GAL4 by heat shocking
- ex. increase T to make GAL80TS stop inhibiting -> activates GAL4
explain: GAL4-UAS for manipulating neurons
- UAS fuse to toxin ex. tetanus (TNT)
⤷ wherever GAL4 expresses, TNT responds
⤷ silences neurons from firing - can also activate neurons ex. optogenetics
explain: optogenetics
- forcing expression of light sensitive receptor in neurons
- ex. channel rhodopsin 2
- excites neurons in the presence of blue light
