Conditional Knockout of genes and the Morris water maze

Question 0

describe the glutamate ionotropic receptors…

Answer 0

mutli subunit ion channels 
activated by ligands 
classified into 3 types based on pharmacology
- NMDA
- AMPA
- Kainate

Question 1

What does the knockout mouse project aim to generate ?

Answer 1

a comprehensive and public resource comprised of mousse embryonic stem cells containing a null mutation in every gene in the mouse genome

Question 2

what is the obligate subunit in NMDA receptors ?

Answer 2

NR1

Question 3

what are the rules of assembly of AMPA receptors ?

Answer 3

homomultimers- 2 or more identical components

heteromultimers- 2 or more different components

Question 4

what are the rules of assembly for kainate receptors ?

Answer 4

GluR5,6 AND 7 are homomultimers

heteromultimers

Question 5

what are the rules of assembly of NMDA receptors ?

Answer 5

heteromultimers
NR1 must partners with one or more NR2 subunits
assemble as hetero-tetramersof 2 obligatory NR1 subunits and 2 regionally localised NR2 subunits

Question 6

what binds to the NR1 and NR2 subunits of glutamate receptors ?

Answer 6

NR1 binds the coagonist glycine

NR2 binds glutamate

Question 7

where is the greater expression of NR1 expression in the mouse brain ?

Answer 7

hippocampus

- also relatively large expression in the cortex

Question 8

where are NR2A subunits highly expressed in the mouse brain ?

Answer 8

hippocampus

Question 9

how was an NMDA receptor NR1 knockout produced?

Answer 9

exons of the gene were replaced with a neomycin resistance gene

Question 10

what happened to the KO NR1 mice?

Answer 10

the homozygous KO mice were born so therefore the mutation does not result in embryonic fatality- body weights were normal so it didnt affect in utero development
BUT
10-20 hours after birth the homozygotes were dead or the mother had removed them from the nest

Question 11

what was the KO mices coat colour and what does this indicate ?

Answer 11

flushed and had a reddish colouration like the wild type

- therefore their cardiovascular and respiratory systems are functionaal at least soo after birth

Question 12

what was the difference shown between the normal pups and the mutant pups?

Answer 12

the mutant pups didnt have any milk in their stomachs

Question 13

where was no abnormality detected in the mutant pups?

Answer 13

no abnormality was detected in the electrocardiographic pattern or the respiratory rhythm

Question 14

what was seen in the mutant mice and what was seen several hourse after birth?

Answer 14

mutant animals were severly ataxic- couldnt support their body weight on their hindlimbs therefore they couldnt get milk to feed
increased apnea is seen several hours after birth and cyanosis occurs shortly before death indicating respiratory distress

Question 15

what is cre-loxP recombination?

Answer 15

special type of site specific recombination

- involves targeting a specific sequence of DNA and splicing it with the help of the enzyme cre recombinase

Question 16

what is the cre protein ?

Answer 16

it is a site specific DNA recombinase which catalyses the recombination of DNA between specific sites in the DNA - sites are known as loxP sequences- they contain specific binding sites for cre

Question 17

what can occur if cells have loxP sites and express cre?

Answer 17

recombination event occurs between the loxP sites

- the cre protein cuts the DNA at both the loxP sites

Question 18

what are the 2 types of transgenic mice strains involved in the cre-loxP system ?

Answer 18

loxP-flanked strain: contains loxP sites flanking a critical portion of the target gene

cell-specific cre expressing strains: contains a transgene that expresses cre under control of a specific promoter- used to producce cell specific knockouts
- these are made by transgenic technology

Question 19

what strains of mice are mated in cell specifc cre-loxP mating scheme ?

Answer 19

strain carrying floxed alleles of the gene to be inactivated
x
strain expressing cre recombinase in aa tissue specific manner
= offspring with gene inactivated in cre expressing tissue

Question 20

in the cell specific cre-loxP mating scheme what would happen if the cre was under the control of a widespread promoter?

Answer 20

a general knockout would be produced

Question 21

what are examples of a widespread promoter?

Answer 21

beta-actin

CMV

Question 22

what is Flp?

Answer 22

flippase- recombinase enzyme

Question 23

what is flippase used for ?

Answer 23

it is used to remove neo cassette
the flippase recognition target = 34bp FRT is positioned to flank the neo so it can be removed
then the flippase cuts at this specific site to cause site specific excisional recombination- this is to produce a cleaner allele

Question 24

how was NR1 just KO in he CA1 region of the hippocampus?

Answer 24

by putting the cre transgene under the control of the aCaMK11 promoter becaause its expression is quite specific to the CA1 pyramidal cells

Question 25

what was trying to be tested by KO of the NR1 subunit in the CA1 region of th hippocampus?

Answer 25

to carry out memory and learning studies

Question 26

why are behaviour test used to understand brain function ?

Answer 26

because behaviour is the final output and visible manifestation of the nervous system psychiatric and neurological disorders involve changes in behaviour

Question 27

what are some examples of different mouse behavioural tests ?

Answer 27

``` startle chamber operant chamber cross maze t maze radial arm maze morris water maze these different tests can be used to test different parts of the brain ```

Question 28

who conceived the morris water maze and what is it used to test ?

Answer 28

1981 by proffessor richard morris | used to assay spatial cognition in rodents

Question 29

explain the morris water maze procedure ...

Answer 29

the rodent is placed in a pool of opaque water at room temp in a random start location rodents are trained over several days to find the platform located in the water pool the latency to find the platform is recorded

Question 30

what happens in probe trials ?

Answer 30

the platform is removed and the % of time spent in the quadrant which contained the platform is recorded and compared to the time spent in the other quadrants

Question 31

what needs to be around the pool and how big is the platform ?

Answer 31

visual ques need to be around the pool | the platform is about 0.5cm-1cm from the surface

Question 32

what is tested 24hours after the mice has been trained?

Answer 32

the mices memory the mice are placed back into the pool without the platform and the amount of times the mouse passes over the area where the platform used to be is recorded

Question 33

what is a limitation of the morris water maze?

Answer 33

the mouse has to be able to swim

Question 34

what are the terms used to describe the different search strategies of the mouse in the morris water maze?

Answer 34

- thigmotaxic/wall hugging - random searching - selectively scanning the inner area - focal search of a target quadrant - focal search in an incorrect area of the pool - direct swimming to the platform - cycling behaviour - chaining response - chaining - crossing all possible platform locations

Question 35

what do the probe trial performances measure ?

Answer 35

they are occupancy based tests to asses the amount of time the animal spends in a virtual area 1) percent quadrant time 2) percent zone 3) crossings 4) proximity measure

Question 36

what is the percent quadrant time?

Answer 36

it is the amount of time mice searched a virtual quadrant which contained the platform during training

Question 37

what is percent zone?

Answer 37

amount of time the mice searched the virtual target zone within a specified distance radius centred on the location of the platform in 60s

Question 38

what is the crossings measurements ?

Answer 38

this is the number of times the mice cross the exact location of the platform (5cm in radius) during a 60s test - it is a counting based measurement

Question 39

what is the proximity measure ?

Answer 39

also known as gallaghers measure average distance in cm of mice from the centre of the platform location across the 60s test it is an error based measure where the animals average distance from the former platform location is recorded

Question 40

what is the morris water maze sensitive to ?

Answer 40

manipulations of the hippocampus - if he hippocampus is damage then the mice perform poorly in the water maze

Question 41

what happened to spatial learning in CA1-KO mice in the water maze test ?

Answer 41

impaired spatial learning - mice are slower in learning the hidden platform

Question 42

what happens to memory of CA1-KO mice in the water maze test ?

Answer 42

impaired spatial memory- mice spend equal time in each of the quadrants in probe trials whereas normal mice spend significantly longer in the target quadrant