Vertebrate Biology

Question 1

How do you do pronuclear injection?

Answer 1

• start with fertilised mouse egg
  -suction pipette hold egg in place
• glass pipette insert DNA fragment into pronuclei
• cell haploid at this stage
• cell becomes diploid (forms zygote )
• cells cellos into morula
• implant egg cell into pseudo-pregnant mouse
• test pups for trans gene and breed with heterozygous
• get homozygous transgenic mouse

Question 2

What DNA is used in pronuclear injection

Answer 2

Promoters
Introns
Polyadenylation sites

Question 3

What are the problems with pronuclear injection

Answer 3

1. Only add gene
2. Expression varies
3. Random insertion

Question 4

What is Homologous recombination?

Answer 4

Swapping over of DNA strands

Question 5

What are embryonic stem cells

Answer 5

Cells that develop earliest in cells - flexible

Question 6

What are the advantages of embryonic stem cells

Answer 6

1. Develop into any cell type
2. Easy to isolate
3. High rate of HR

Question 7

What’s a knock in gene

Answer 7

Insert gene into specific position

Question 8

What’s a knockout gene

Answer 8

Inactive gene by using a target vector
E.g NeoR region into chromosome and into functional gene to survive AB exposure

Question 9

How do you make a knockout mouse

Answer 9

• target vector into cells by liquid culture
• electroporation: open channels in membrane DNA into cell
  -2 rounds of selection
  -use fur colour as mark for correct insertion

Question 10

How was dolly the sheep made

Answer 10

• insert nucleus from animal want to grow
• fully differentiated cells taken from mammary glad of adult sheep and grown in culture
  -starve cells
  -electric current to die cell with empty egg cells and nucleus
  -cell behave like newly fertilised cell
  -implant in surrogate - birth
• diff face colour if work

Question 11

How was first transgenic fish made ?

Answer 11

Goldfish express human GH, done so by injecting DNA into fertilised egg cytoplasm as couldn’t see nuclei

Question 12

How is AquAdvantage salmon made

Answer 12

-ocean pout antifreeze protein gene (APS), 5’ promoter r and 3’ terminator link to salmon GH
-promoter allow GH production outside of pituitary gland- all season
- reach market size in 1 year

Question 13

AquAdvantage sustainability claims

Answer 13

1. Recycle water to prevent disease and polluiton
2. Inland so no wild type mating
3. Green closer to consumer so less CO2

Question 14

Why is xenotransplantation important/ how done

Answer 14

Pig organs used for humans.
Pug organs have carb antigen on causing immune response from humans
Knockout alpha 1.3 galatosyl transferase using HR