BIO2015: Lecture 6

After embryonic cells have been selected, how does one tell if they have survived after injection?

Inject ES cells from brown fur mouse; use blastocyst from black fur mouse - look at the coat colour of the pups

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what is a chimera?

mixture of cells from more than one animal

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what is a gene knockout?

a genetic technique in which one of an organism's genes are made inoperative

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when are gene knockouts used?

in learning about a gene that has been sequenced, but which has an unknown or incompletely known function

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what is an example of a gene knockout that has been studied in mice?

Melanocortin-3 receptor- An important regulator of body fat storage- over 10,000 gene knock-outs described in mice

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what is an example of the application of the pronuclear injection technique?

farm animals as ‘bioreactors'- ‘Pharming’ – pharmaceuticals from farm animals - main application for transgenesis- Potential to replace recombinant bacteria and cell cultures

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what are the advantages of pharming?

Large scale production, low running costs, correct glycosylation and post-translational modification of proteins

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who was 'Tracy'?

first transgenic sheep to produce human protein in milk (1991)

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what was PPL therapeutics a spin off from?

Roslin institute (1987)

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what was Human α1-antitrypsin (AAT) a treatment for?

cystic fibrosis lung disease

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in farm animals, what is the integration efficiency for pronuclear injection?

1-5% (5-40% in mice)- very inefficient

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is it possible to isolate stem cells from farm animals?

no

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What was the aim of the Dolly research?

To provide an alternative in livestock to mouse embryonic stem cells

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define differentiation

Developmental events between fertilised zygote and mature organism- Specialisation of cells to fulfill specific functions e.g. kidney cell, neurone, etc.

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what was the key development in the dolly research?

Cultured cells induced by starvation to stop growing and enter quiescent (G0) phase of cell cycle - become ‘re-programmable’ when nucleus injected into metaphase II egg

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what was the process of cloning dolly?

full differentiated cell from mammary gland, grown in culture was deprived of nutrients- starvation induced quiescent stage this was fused by electrical shock to an enucleate egg cell to produce egg cytoplasm containing donor cell nucleus

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what did this egg/nucleus fusion mature into?

embryo

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how did Dolly die?

Died of a respiratory infection unrelated to cloning, but gave birth to normal lambs, developed premature arthritis (linked to cloning?)

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what is an example of naturally occurring reproductive cloning?

identical twins

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how else can reproductive cloning occur?

embryo splitting

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is cloning by nuclear transfer transgenesis or not?

no

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how can nuclear transfer be applied to livestock?

genetic improvement- allows rapid introduction of genes from elite animals- can also be used for conservation of endangered breeds of animals

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what is the impact of nuclear transfer on making transgenic farm animals?

nuclear transfer allows mouse stem cell methods to be used, providing precise gene targeting – second generation of transgenic livestock More sophisticated approaches possible than first generation of transgenic livestock by pronuclear injection

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BIO2015: Lecture 6

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