manipulating cells in culture; generation of in vivo gain-of-function and loss-of-function models

Question 1

what is loss of function?

Answer 1

reduced activity
- in heterozygous state to half normal levels of the protein product or complete loss of the gene product

Question 2

what is gain of function?

Answer 2

increased levels of gene expression
- development of a new function of the gene product

Question 3

how is gene silencing achieved?

Answer 3

• RNA interferance (RNAi)
• CRISPR/Cas9

Question 4

how is gene overexpression achieved?

Answer 4

• expression vectors
• CRISPR/Cas9

Question 5

siRNA (small interefering RNA)

Answer 5

• chemically synthesized
• consists of two RNA strands (sense and antisense)
• transfection to the cytosol
• siRNA integration to the RISC (RNA induced silencing complex)
• strand separation within RISC
• antisense strand hybridises to the complementary (target) mRNA
• cleavage of targeted mRNA within RISC
• further degradation by other endogenous nucleases
• transient silencing

Question 6

short hairpin RNA (shRNA)

Answer 6

• synthesized within the cell, two RNA strands linked by a short loop
• DNA plasmid transfection or lentiviral transduction
• transcription, export to cytosol
• DICER removes the loop (siRNA production), siRNA loading to RISC, removal of one RNA strand
• target mRNA with complementary sequence, cleavage of mRNA + further degradation
• stable knock-down cell lines

Question 7

three variations of CRISPR

Answer 7

• genome engineering with Cas9 nuclease
• genome engineering by double nicking with paired Cas9 Nickases (= HDR)
• localisation with defectie Cas9 nuclease

Question 8

why do we carry out expression vectors to trigger gene overexpression?

Answer 8

• assess effect of having a protein/RNA of interes in excess
• overexpress mutant protein
• recover WT phenotype in mutant/defective background
• study function of heterologous proteins
• analysis of interacting molecules (co-immunoprecipitation)

Question 9

what are the five types of vectors/plasmids?

Answer 9

• cloning plasmids
• expression plasmids
• gene-knock down plasmids
• reporter plasmids
• viral plasmids

Question 10

what are cloning plasmids?

Answer 10

facilitate cloning of DNA fragments
- bacterial resistance gene, origin and MRS

Question 11

what are expression plasmids?

Answer 11

used for gene expression
- promoter, transcription terminator sequence, inserted gene

Question 12

what are gene knockdown palsmids?

Answer 12

reduce expression of endogenous gene
- shRNAm promoter for expression of short RNAs

Question 13

what are reporter plasmids?

Answer 13

study the function of the genetic elements
- promoter, reporter gene (Luciferase, GFP)

Question 14

what are viral plasmids?

Answer 14

deliver genetic material into target cells

Question 15

where are epitope tags found?

Answer 15

carried by two commonly used mammalian expression vectors
- usually in 5’ region after MCS (multiple cloning site)

Question 16

what are epitope tags?

Answer 16

method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques.
- fusion gene cloned into an appropriate expression vector for the experimental cell type and host cells are transfected

Question 17

GFP (green fluorescent protein)

Answer 17

• naturally produced in jellyfish; Aequorea victoria
• discovered in 1960s
• source of bioluminescence when exposed to UV light

Question 18

why is using GFP useful?

Answer 18

• tags cells and helps us trace to a target protein
• acts a reporter gene, link it to another gene to show if it is expressed

Question 19

what is site-directed mutagenesis?

Answer 19

introduces point mutations
- a method to create specific, targeted changes in double stranded plasmid DNA.
- used to study changes in protein activity that occur as a result of the DNA manipulation.

Question 20

process of site directed mutagenesis

Answer 20

1. mutant strand synthesis done by denaturing DNA template, annealing primers with desired mutation (1hour)
2. Dpn I digestion of template , digestion of parental methylated and hemimethylates DNA with new Dpn I enzyme (5mins)
3. transform mutated molecules into competent cells for nick repair (1.5hours)

Question 21

methods for DNA and RNAi delivery; chemical transfection

Answer 21

• uses ; calcium phosphate, cationic polymers, lipofection, - transcient/stable transfection
• suitable for sensitive primary cells

Question 22

methods for DNA and RNAi delivery; physical transfection

Answer 22

• microinjection
• electroporation (nucleofection-primary cells)
• transient/stable transfection
• not suitable for sensitive primary cells

Question 23

what does transient transfection mean?

Answer 23

expression of foreign DNA for a limited time (24-96hours)
- NO DNA INTEGRATION

Question 24

what is a stable transfection?

Answer 24

integration of foreign DNA in the genome of the cells

Question 25

summary of manipulating genes in culture

Answer 25

• generating gain or loss of function in vitro models to study the functional impact of genetic variants
• different types of plasmids used for different purposes; from DNA cloning to protein expression
• expression vectors can contain tags e.g. GFP
• different cell transfection methods can be used to introduce genetic material inside cells in culture, they can be chemical or physical and trasnfection can be transient or stable
• site directed mutagenesis is used to introduce mutations in expression plasmid
• siRNA and shRNA are short RNA molecules that when introduced in host cell can downregulate gene expression by targeting mRNA in transient or more stable way.