experimental methods (dr maloney) Flashcards
steps of forward genetics/forward genetic screens
- mutate LOTS of genes (either w/radiation or CRISPR)
- find the animals that show a specific phenotype (whatever you are looking for)
- sequence genome and see which gene was mutated/knocked out
what is it called when you start with a population and mutate a bunch then look for phenotype and see which gene was mutated/knocked out
forward genetic screen
explain the steps of a reverse genetic screen/reverse genetics
- start with gene of interest
- see if there is an effect/what the effect is when that gene is removed
what is it called when you start with a gene of interest and then see what happens when you remove it
reverse genetic screen
using CRISPR to knock out genes you need
- guide RNA for gene of interest
- Cas9 protein
using CRISPR to add gene(s) you need
- guide RNA for gene of interest
- Cas9 protein
- DNA of protein you want to add
how can you use viruses to alter genes
- AAVs add a gene
- rabies/pseudorabies viruses infect presynaptic cells and allows us to know what is providing info/input to cells
what to optogenetics do (general)
effect protein activity
what do consititutive activity silencers do (general)
affect protein activity
what do chemogenetics do (general)
effect protein activity
what do thermogenetics do (general)
effect protein activity
what substances are used in optogenetics
- channelrhodopsin (+)
- halorhodopsin (-)
what substances are constitutive activity silencers
- inward rectifying K+ channels (-)
- tetanus (-)
what substances are used in chemogenetics
DREADs (+ or -)
what is chemogenetics
altering activity by injecting chemicals
what is optogenetics
using light to alter activity
substances used in thermogenetics
- shibire (-)
- TRP channels (+)
what is thermogenetics
using tempurature to alter activity (only in non-mammals)
proteins that let you LOOK at activity
gCAMP (calcium sensor)
proteins that let you see where neurons are
GFP (green fluorescent protein) and derivatives
proteins that let you see who they are connected with
- GRASP
- trans-tango
and rabies/pseudorabies but those arnt proteins, rather another method
steps to use CRISPR
- identify target gene sequence
- create guide RNA specific to target sequence
- guide RNA attached to Cas9 protein and introduced into target cells
- locates target DNA sequence and Cas9 cuts DNA
- scientists can alter DNA sequence (deletions, additions, swapping)
what do binary expression systems allow scientists to do/what is the point
allows scientists to:
- look at spaciotemporal patters on genes of interest
- look at role of specific protien function(s) through upregulation and knockout
explain the ‘driver’ line of flies in binary expression systems
driver line: has a DNA insertion that encodes for a transactivator
transactivator activates the expression of another gene
enhancer controls the expression of the transactivator —- only active in certain cells at certain times
explain the ‘reporter/effector’ line of flies in binary expression systems
reporter line: has a DNA insertion containing a reporter gene
reporter has an activation sequence and is only expressed when transactivator binds to the reporter activation sequence
after crossing the ‘driver’ line and ‘reporter’ line, the offspring contain both sets of genes. what is the outcome when enhanver is active vs inactive
enhancer active = transactivator and reporter expressed
enhancer inactive = transactivator and reporter NOT expressed
what is a common reporter
GFP (green florecent protein)
when enhancer is active = cells glow green
