Molecular Approaches to Studying NS Development

Question 1

What is the purpose of in situ hybridisation?

Answer 1

Detect RNA expression in embryo

Question 2

Describe the process of in situ hybridisation

Answer 2

Antisense probe to RNA labelled with DIG - binds were RNA expressed
Anti-DIG antibody binds to DIG probe
Antibody conjugated to fluorescent molecule/alkaline phosphatase (blue)

Question 3

What is the purpose of immunofluorescence?

Answer 3

Detect proteins

Question 4

Describe the process of immunofluorescence

Answer 4

Primary antibody against species-specific antigens on protein
Secondary antibody coupled to fluorescent molecule - against primary antibody

Question 5

What does loss of function analysis show?

Answer 5

Gene required

Question 6

What does gain of function analysis show?

Answer 6

Gene sufficient

Question 7

What are the disadvantages of murine gene KO studies?

Answer 7

Some developmentally lethal - no adult studies
May fail to produce observable change
May produce different change to in humans

Question 8

Describe the process of forming chimaeric mice

Answer 8

Inject ES cells with target mutation into mouse blastocyst
Implant in surrogate mother
Forms chimaeric offspring

Question 9

What is the purpose of the Cre-lox system?

Answer 9

Time/region specific gene KO

Question 10

Describe the process of the Cre-lox system

Answer 10

Cre recombinase binds loxP sites - homologous combination of target gene between - DNA excision

Question 11

Describe the process of gain of function transgenic mouse creation

Answer 11

Design vector with cDNA for chosen gene
Pronuclear injection - random integration into genome
Identify carriers of transgene

Question 12

Describe the process of gene overexpression

Answer 12

Inject DNA into neural tube lumen
Electroporate
Visualise changes with GFP after 24 hours

Question 13

Describe the process of morpholino antisense oligonucleotides

Answer 13

Short antisense oligonucleotides specific to target mRNA
Modified bases have morpholino ring - stable
Targets 5’-UTR of mRNA / coding region near translation start site
Inject into cell/embryo
Blocks translation
Measure loss of expression at protein level - using antibody

Question 14

What is the purpose of RNA interference?

Answer 14

KO expression of certain protein in cell line

Recently KO protein for multiple generations

Question 15

Describe the process of RNA interference

Answer 15

Short dsRNAs complementary to target mRNAs - siRNAs
siRNAs incorporated into protein complexes - RNA-induced silencing complexes - RISCs - strands separated
One strand guides RISC to complementary mRNA
Argonaute enzyme catalytic component of of RISC - destroys targeted mRNA

Question 16

What are the purposes of viral vectors?

Answer 16

Increase/decrease endogenous protein levels

Introduce novel proteins

Question 17

What are the advantages of viral vectors?

Answer 17

In vivo / in vitro
Expression lasts weeks/months
Directed to certain brain area
Any animal can be used - not inbred like transgenic animals
Cheaper than transgenic animals
Do not replicate - safe and easy to use

Question 18

What is the use of a lentivirus vector?

Answer 18

Transgene integrates into host DNA
Gives long-lasting expression
Transgene passed to progeny

Question 19

What is the use of an adeno-associated virus vector?

Answer 19

Transgene remains extrachromosomal - episomal

Transgene eliminated

Question 20

Which viral vector type has a larger capacity?

Answer 20

Lentivirus

Question 21

What are the disadvantages of viral vectors?

Answer 21

Inject every animal - time-consuming
Cannot get global effect - virus does not spread around whole body
Cannot place dangerous constructs into viral vectors