experimental methods (dr maloney)

Question 1

steps of forward genetics/forward genetic screens

Answer 1

1. mutate LOTS of genes (either w/radiation or CRISPR)
2. find the animals that show a specific phenotype (whatever you are looking for)
3. sequence genome and see which gene was mutated/knocked out

Question 2

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

Answer 2

forward genetic screen

Question 3

explain the steps of a reverse genetic screen/reverse genetics

Answer 3

1. start with gene of interest
2. see if there is an effect/what the effect is when that gene is removed

Question 4

what is it called when you start with a gene of interest and then see what happens when you remove it

Answer 4

reverse genetic screen

Question 5

using CRISPR to knock out genes you need

Answer 5

1. guide RNA for gene of interest
2. Cas9 protein

Question 6

using CRISPR to add gene(s) you need

Answer 6

1. guide RNA for gene of interest
2. Cas9 protein
3. DNA of protein you want to add

Question 7

how can you use viruses to alter genes

Answer 7

1. AAVs add a gene
2. rabies/pseudorabies viruses infect presynaptic cells and allows us to know what is providing info/input to cells

Question 8

what to optogenetics do (general)

Answer 8

effect protein activity

Question 9

what do consititutive activity silencers do (general)

Answer 9

affect protein activity

Question 10

what do chemogenetics do (general)

Answer 10

effect protein activity

Question 11

what do thermogenetics do (general)

Answer 11

effect protein activity

Question 12

what substances are used in optogenetics

Answer 12

1. channelrhodopsin (+)
2. halorhodopsin (-)

Question 13

what substances are constitutive activity silencers

Answer 13

1. inward rectifying K+ channels (-)
2. tetanus (-)

Question 14

what substances are used in chemogenetics

Answer 14

DREADs (+ or -)

Question 15

what is chemogenetics

Answer 15

altering activity by injecting chemicals

Question 16

what is optogenetics

Answer 16

using light to alter activity

Question 17

substances used in thermogenetics

Answer 17

1. shibire (-)
2. TRP channels (+)

Question 18

what is thermogenetics

Answer 18

using tempurature to alter activity (only in non-mammals)

Question 19

proteins that let you LOOK at activity

Answer 19

gCAMP (calcium sensor)

Question 20

proteins that let you see where neurons are

Answer 20

GFP (green fluorescent protein) and derivatives

Question 21

proteins that let you see who they are connected with

Answer 21

1. GRASP
2. trans-tango

and rabies/pseudorabies but those arnt proteins, rather another method

Question 22

steps to use CRISPR

Answer 22

1. identify target gene sequence
2. create guide RNA specific to target sequence
3. guide RNA attached to Cas9 protein and introduced into target cells
4. locates target DNA sequence and Cas9 cuts DNA
5. scientists can alter DNA sequence (deletions, additions, swapping)

Question 23

what do binary expression systems allow scientists to do/what is the point

Answer 23

allows scientists to:

1. look at spaciotemporal patters on genes of interest
2. look at role of specific protien function(s) through upregulation and knockout

Question 24

explain the ‘driver’ line of flies in binary expression systems

Answer 24

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

Question 25

explain the ‘reporter/effector’ line of flies in binary expression systems

Answer 25

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

Question 26

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

Answer 26

enhancer active = transactivator and reporter expressed

enhancer inactive = transactivator and reporter NOT expressed

Question 27

what is a common reporter

Answer 27

GFP (green florecent protein)

when enhancer is active = cells glow green