Reverse Genetics

Question 1

Give the definition of reverse genetics.

Answer 1

Reverse genetics = seeks to find the phenotypes linked to specific sequences of DNA.

Question 2

Give the process of reverse genetics.

Answer 2

1. Alter gene in vitro
   -get altered sequence synthesised or use recombinant DNA techniques
2. Introduce into cell
   -direct uptake
   Incubate DNA with competent cells
   Bacterial/yeast transformation
   Animal cell transfection

-aid/induce uptake
Electroporation
Microinjection
Virus-mediated
Ballistic (gene gun or cells with walls)
Agrobacterium tumefaciens-mediated (plants and some fungi)

-fate of the transgene
Transient expression
Replicates on a plasmid
Chromosomal integration (random or targeted to a particular locus)

-cell type
Somatic or germ
Haploid or diploid

-detection of transgenic cells
Selectable marker gene
Yeast & bacteria often use auxotrophic mutants

3. Determine phenotypic effects

Question 3

Describe how CRISPR is used for gene editing.

Answer 3

• CRISPR-mediated viral defence in bacteria
• bacteria cleave off small sequence from DNA of an infecting virus that they have destroyed and integrate into their own bacterial genome
• this DNA is transcribed from the CRISPR locus, processed and bound to Cas protein
• easily altered guide RNAs can guide the Cas9 endonuclease to a specific gene

Question 4

Describe promoter activity reporters & limitations of using them.

Answer 4

• clone reporter after promoter sequence, replacing native ORF Level detected should reflect promoter activity
• most commonly used is Green flurorescent protein (GFP)
• runs into problems with haploid organisms, complex promoters, or organisms with no formal genetics

Question 5

Give the purpose of protein localisation & movement.

Answer 5

Tag protein with GFP so when it is incorporated it will fluoresce and so we can see it.

Question 6

Describe the process of RNA interference.

Answer 6

• introduce double strands of DNA into cells, they become incorporated into argonaute or Piwi proteins to target RNAs produced by cell
• specific knockdown of gene expression

Question 7

Why is RNAi used as experimental tool for specific gene knockdown?

Answer 7

• applicable to many eukaryotes

- individual gene knockdown

Question 8

Why are high throughput screens required?

Answer 8

Organisms are complex.