lecture 20

Question 1

What is transgenesis?

Answer 1

the process of transferring genes to animals

transgenics refers to a gain of function

Question 2

What is a transgenic animal?

Answer 2

an animal that carries a transgene

Question 3

What is a transgene?

Answer 3

the foreign gene

Question 4

What do you start with if you are trying to make a whole animal with a mutation? Why is that significant?

Answer 4

fertilised egg

• supposedly male pro-nucleus is slightly larger than female pro-nucleus
• haploid nucleus of the male has been transferred by fertilization into the centre of the egg
• female egg of all mammals is frozen at metaphase II of meiosis
• half extruded in a polar body
• two halves of the nucleus join, create a spindle and then undergo cell division to create two cells
• if you inject DNA/RNA into one of the haploid genomes when they mount up on the spindle to undergo their first cell division every cell after that will have the DNA
• if you miss that time point, e.g. into one cell of a two cell embryo, only half will have the insert
• therefore critical to be injected into a haploid genome
• before spindling

Question 5

What is the transgenic technique?

Answer 5

• mate mice
• flush out fertilised eggs from oviduct
• have about 12 hours in mice
• inject transgene construct into one pronucleus
• culture blastocysts
• implant into pseudopregnant host female
• prepare genomic DNA from tails of offspring
• analyse for presence of transgene e.g. by PCR analysis

Question 6

What are properties of the transgene?

Answer 6

• inject picolitres of the gene construct - not much, but each has many copies of the gene
• insert into only one site in the genome
• the transgene randomly integrates into the host cell genome
• transgene can become stably integrated into the germ line
• once you’ve created the animal you can create a line of animals
• can freeze down sperm of the animals

Question 7

What is random integration of the transgene?

Answer 7

• the transgene can integrate anywhere in the genome
• if it integrates into the promoter region of another gene it will interrupt the function of the gene: increase, decrease or no effect of transgene expression may lead to cell proliferation, apoptosis etc
• if it integrates into gene it may generate a defective protein, may lead to cell proliferation, apoptosis etc
• all of this can lead to insertional mutagenesis: generation of mutations in DNA by insertion of one or more bases

Question 8

How do we get tissue specific gene expression?

Answer 8

transgene construct: e.g. rat insulin promoter + growth factor

sometimes these promoters are leaky because you aren’t taking the full promoter

Question 9

What is pharming for farmaceuticals?

Answer 9

• pronuclear injection current method e.g. pigs, rabbits, goats, cows, rats, mice, sheep
• transgene directed to mammary gland, blood cells, bladder cells. Milk, blood, urine (or egg white in chicks)
• large daily output, post-translational processing, low capital cost, easy access to transgene protein
• mammary gland promoters: beta-lactoglobulin, beta-casein, alpha s 1-casein, whey acidic protein
• generate transgenic construct: beta-lactoglobulin + transgene
• inject into fertilised egges
• generate line of sheep expressing transgene protein into milk
• collect and fractionate milk
• purify transgene protein

examples of human proteins secreted into milk of transgenic animals =

• antithrombin III in goat for thrombosis (GTC biotherapeutics USA)
• tPA in goat for thrombosis (PPL therapeutics UK)
• A-antitrypsin in sheep for emphysema (“ “)
• Factor IX in sheep for haemophilia (“ “ )
• polyclonal antibodies in cattle for vaccines (hematech USA)

animals as bioreactors

Question 10

How can we use an animal model of human disease?

Answer 10

interleukin-6 (IL-6)

• important cytokine for blood development and immune system function
• IL-6 levels are high in inflammation, infection, or trauma induced disorders of the central nervous system

to understand function of IL-6:

• generate a transgenic animal that over-expresses IL-6 in the brain (animal model)
• transgene injected into pronucleus (brain-specific promoter and IL-6 cDNA)
• result:
• • animals have nerver damage – severity correlates to level of IL-6
• • this model demonstrates that IL-6 has a role in the pathology of degeneration in the brain

Question 11

How can we use transgenics to improve livestock?

Answer 11

Selective breeding has been done for centuries
transgenics fast tracks this process

e.g. transgenics that over-express growth hormone vs selectively breeding naturally big pigs

results:
- increase in rate of growth
- decreased amount of feed required per unit weight gain
- reduced levels of body fat lipids, cholesterol, lower saturated and unsaturated fats
(pigs normally only have growth hormone for two months)
also:
- reduced reproductive performance
- arthritis
- gastric ulcers
- renal disease
- premature death

these technologies are good but they have dark and light sides

• be informed
• just printing the good side is not a fair assessment

Question 12

What are disadvantages of using animals as bioreactors?

Answer 12

1. unexpected or undesired effects of the transgene
   - leaky expression
2. effect of the transgene on the health of the animal

Question 13

How do transgenics compare to gene targeting?

Answer 13

transgenics vs gene-targeted

• technique to transfer transgene: pronuclear injection vs electroporation
• integration type: random vs site-specific/targeted
• is foster mother required?: yes – grow to blastocyst stage, transfer to host uterus, one generation for effect of transgene vs yes – inject targeted cells into host blastocyst, transfer to host uterus. Breed +/- to generate -/-. Analyse phenotype of -/-
• detect transgene: DNA analysis (both)
• germline?: yes (both)
• species: all vertebrates vs mice

Question 14

How do the efficiencies of different techniques compare?

Answer 14

• transgenic: 60-80%, relies on construct integrating randomly in the zygote
• conventional gene-targeting: 1-6%, homologous recombination, ES cells
• zinc finger nucleases: 1-20%, DNA mis-repair, zygote
• CRISPR/Cas system: 1-20%, DNA misrepair, zygote