MNG3701 FINAL PAPER UPDATED
COMPLETE QUESTIONS AND CORRECT
ANSWERS BUNDLED REVIEW GUIDE
●● uses to TM
Answer: -mark specific organelles
-trace axons and dendrites with membrane bound fluorescent reporters
-put in genes measuring neural activity (Ex: detect
ca, voltage, vesicle fusion)
-introduce genes to induce/inhibit neural activity (Ex: channel
rhodopsinis, halorhodopsins, DREADDS)
-ablate/kill cells by expressing toxin or toxin receptors
●● making TM
Answer: -transgene = what is added to specific cells
-inject into eggs (new gene can be human or mouse)
-implant in pseudopregnant mouse (she is given hormones to be able to
carry eggs to term)
-has babies, then PCR ran (shows which have new gene)
-use those to breed and experiment
●● knockout/knockin mice
,Answer: Why:
KO
-inactivate gene (look at function)
-inactivate gene in certain cells
-inactivate at certain time in development (if early activation = lethal)
KI
-can replace with modified gene (determined effect in disease gene)
Construction:
-create targeting construct
-place in ES cells
-select for proper incorporation
-place ES --> blastocyst
-place blastocyst --> pseudopregnant mouse
-breed
●● general TM strategy
Answer: 1. use stem cells --> blastocyst
2. construct targeting vector
-contains pieces homologous to the target & new inserted portions
-goes to specific locations
-homologous - new -homologous - HSV-tk
,3. transfection
-HSV-tk = lost if done correctly
-new (neo) is like a bubble that gets added in
4. proliferation
-only small number of cells get the new (neo) gene
-positive-negative selection (kill cells that do NOT have new gene to
isolate new ES cells)
-selects for yes neo, no HSV-tk
5. inject ES --> blastocyst
-this makes a mosaic (both types of cells)
-implant into pseudomother
6. breeding
-mosaic babies + normal babies = normal or mutated (no mosaic)
-check if successful : do tail snips then run gel & compare
●● knock out
Answer: -disrupt DNA
-can be added at center or end
●● knock in
Answer: -doesn't disrupt
-only adds at end
, ●● Cre-Lox system
Answer: -KO gene at specific time or in specific place
-tissue specific promoter (Cre Recombinase) + Lox before and after
targeted gene = gene cut out between lox for specific spot/time (at the
site of Cre)
Place
-aka conditional knockout
-LoxP + Promoter & Cre
-Cre-excised in promoted & not excised in others
Time
-aka inducible knockout
-LoxP + CreER & Promoter
-LoxP in promoted and not others (Cre= inactive) + 4-hydroxytamoxifen
(makes Cre active) = Cre-excised in promoted
●● CRISPR-Cas9
Answer: -CRISPRs = clustered regularly interspaced palindromic
sequences
-array of identical repeats intercalated with DNA - targeting spacers
derived from bacteria and plasmids
COMPLETE QUESTIONS AND CORRECT
ANSWERS BUNDLED REVIEW GUIDE
●● uses to TM
Answer: -mark specific organelles
-trace axons and dendrites with membrane bound fluorescent reporters
-put in genes measuring neural activity (Ex: detect
ca, voltage, vesicle fusion)
-introduce genes to induce/inhibit neural activity (Ex: channel
rhodopsinis, halorhodopsins, DREADDS)
-ablate/kill cells by expressing toxin or toxin receptors
●● making TM
Answer: -transgene = what is added to specific cells
-inject into eggs (new gene can be human or mouse)
-implant in pseudopregnant mouse (she is given hormones to be able to
carry eggs to term)
-has babies, then PCR ran (shows which have new gene)
-use those to breed and experiment
●● knockout/knockin mice
,Answer: Why:
KO
-inactivate gene (look at function)
-inactivate gene in certain cells
-inactivate at certain time in development (if early activation = lethal)
KI
-can replace with modified gene (determined effect in disease gene)
Construction:
-create targeting construct
-place in ES cells
-select for proper incorporation
-place ES --> blastocyst
-place blastocyst --> pseudopregnant mouse
-breed
●● general TM strategy
Answer: 1. use stem cells --> blastocyst
2. construct targeting vector
-contains pieces homologous to the target & new inserted portions
-goes to specific locations
-homologous - new -homologous - HSV-tk
,3. transfection
-HSV-tk = lost if done correctly
-new (neo) is like a bubble that gets added in
4. proliferation
-only small number of cells get the new (neo) gene
-positive-negative selection (kill cells that do NOT have new gene to
isolate new ES cells)
-selects for yes neo, no HSV-tk
5. inject ES --> blastocyst
-this makes a mosaic (both types of cells)
-implant into pseudomother
6. breeding
-mosaic babies + normal babies = normal or mutated (no mosaic)
-check if successful : do tail snips then run gel & compare
●● knock out
Answer: -disrupt DNA
-can be added at center or end
●● knock in
Answer: -doesn't disrupt
-only adds at end
, ●● Cre-Lox system
Answer: -KO gene at specific time or in specific place
-tissue specific promoter (Cre Recombinase) + Lox before and after
targeted gene = gene cut out between lox for specific spot/time (at the
site of Cre)
Place
-aka conditional knockout
-LoxP + Promoter & Cre
-Cre-excised in promoted & not excised in others
Time
-aka inducible knockout
-LoxP + CreER & Promoter
-LoxP in promoted and not others (Cre= inactive) + 4-hydroxytamoxifen
(makes Cre active) = Cre-excised in promoted
●● CRISPR-Cas9
Answer: -CRISPRs = clustered regularly interspaced palindromic
sequences
-array of identical repeats intercalated with DNA - targeting spacers
derived from bacteria and plasmids
