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PHRM3041 > Biotechnology 1 - Transgenic/knockout animals > Flashcards

Biotechnology 1 - Transgenic/knockout animals Flashcards

1
Q

Biotechnology use in…

A

drug discovery - disease mechanism
(drug lead -HTS, target identification -microarray)

Clinical (Tx - monoclonal antibodies, Diagonosis)

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2
Q

What can you produce with biotechnology

A

produce recombinant proteins and peptides

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3
Q

Biotechnology tools

A

Transgenic and knockout

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4
Q

What are transgenic animals?

A

Any animal into which cloned genetic material HAS BEEN TRANSFERRED

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5
Q

What are knockout animals?

A

Any animal in which a GENE PRODUCT HAS BEEN RENDERED deficient

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6
Q

Similarity between transgenic and knockout animals

A

change is an inheritable trait and is passed onto successive generations

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7
Q

What are transgenic animals used for?

A
  1. models of disease to aid research (overexpression of tumour promoters)
  2. models to aid metabolism testing (overexpression of P450 enzyme)
  3. molecular pharming (produce human proteins and other pharmaceuticals in LARGE QUANTITIES) eg. pigs and cows that prdocue proteins that dissolve blood clots
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8
Q

What are knockout animals used for?

A
  1. models of disease to aid research (cancer, parkinson’s disease - knockout of genes)
  2. models to understand human systems (knockout a gene to determine function of the gene or the effect of drug that targets the gene–> specificity and selectivity of drugs)
  3. assess tissue-specific pharmacology and physiology - receptors/channels
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9
Q

Conditional knockout animals are…

A

gene product knockout in a specific tissue

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10
Q

How to make transgenic mouse?

A
  1. pronucleus injection

2. blastocyst injection

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11
Q

What is pronucleus injection?

A

Injecting desired gene to pronucleus of fertilised mouse egg

DNA into pronucleus

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12
Q

What is blastocyst injection?

A

transforming embryonic stem cells with desired DNA then injecting it into blastocyst

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13
Q

Pronucleus injection METHODS

A
  1. Make transgene (coding sequence + promoter + enhancer sequence) by using recombinant DNA methods
  2. Incorporate transgene into a vector (or plasmid)
  3. Remove fertilised eggs from female mouse
  4. Inject transgene into MALE pronucleus
  5. Implant oocytes into foster female and develop normally
  6. Genotyping - do PCR analysis using tissue from the tail to determine if the offspring carries the transgene
  7. Establish a transgenic strain by mating the offsprings to make homozygous
    (ones found in step 6 are heterozygous)
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14
Q

In pronucleus injection, why is transgene injected into male egg?

A

it is more successful

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15
Q

Survival rate of microinjection and implantation in pronucleus injection

A

very low, 10-30% survive the injection,

only 40% carry the transgene

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16
Q

gene and expression in pronucleus injection

A

we can see the gene everywhere in the body, but expression at specific parts

17
Q

Advantage of pronucleus injection

A

applicable to most animals

all offspring will be heterozygous because we injected the transgene directly into the eggs

18
Q

Disadvanage of pronucelus injection

A

DNA incorporates into the genome randomly (can knock out wrong cells), so it is important to screen
Low efficiency/success rate

19
Q

What is a stem cell?

A

pluripotent cell capable of becoming any cell

20
Q

Difference between blastocyst and blastocyte

A

Blastocyst is a hollow ball of cells made of outer trophoblast cells and inner cell mass
Blastocyte is just undifferentiated embryonic cell

21
Q

When is ES cell harvested

A

from 3.5 day embryos

22
Q

Blastocyst injection METHODS

A
  1. Make the transgene
  2. Isolate the ES cells and culture them on petri dish on a feeder layer of fibroblasts (so they proliferate and can be cultured continously)
  3. Expose ES cell to transgene by infection/transfection
  4. Select for successfully transformed cells on the dish (eg. antibiotic environment)
  5. Inject the ES cell to blastocyst of different characteristics mouse to make a blastocyst with both genes (original and new mouse)
  6. Reimplant it into the foster female and allow develop normally
  7. Select the chimera, establish the strain by mating 2 heterozygous to make homozygous (transgene in the germline)
23
Q

What is germline?

A

A series of germ cells each descended or developed from earlier cells in the series

24
Q

Why isn’t chimera or partially transgenic mice good enough?

A

because they are just mouses with heterozygous genes, we want them to have it homozygous

25
Q

Disadvantage of transgenic methods

A

Large cost and scale

random insertion of transgene into chromosomes –> we do not know where it is and it could knock out another gene

26
Q

how to make knockout animals

A

we need to specifically target this gene

27
Q

Only viable method to make knockout animal is?

A

blastocyst injuection because it allows us to target a specific gene

28
Q

How do we target specific gene in knockout animal production?

A

Homologous recombination

29
Q

What is homologous recombination?

A

crossover occurs in a cell (at meiosis) between identical or very similar DNA sequences –> chiasma
This is very rare in pronucelus injection
We can increase this event by ensuring high similarity between transgene and target gene (use both mouse)

30
Q

Process of knockout

A
  1. isolate gene to be knocked out from a mouse
  2. Make a new DNA sequence (identical to gene)
  3. Incorporate marker gene (eg. fluorescence)
  4. Put transgene in ES cell and select the good ones
  5. Inject the good ones into blastocyst of foster animal
  6. proceed with blastocyst injection method
31
Q

Issues with transgenic/knockout method

A

Random insertion of transgene
interpretation of a phenotype is not always straightforward
animals do not always mimic humans
transgene may not be expressed to physioloically relevant levels

PHRM3041 (9 decks)

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