Biotechnology 1 - Transgenic/knockout animals Flashcards
Biotechnology use in…
drug discovery - disease mechanism
(drug lead -HTS, target identification -microarray)
Clinical (Tx - monoclonal antibodies, Diagonosis)
What can you produce with biotechnology
produce recombinant proteins and peptides
Biotechnology tools
Transgenic and knockout
What are transgenic animals?
Any animal into which cloned genetic material HAS BEEN TRANSFERRED
What are knockout animals?
Any animal in which a GENE PRODUCT HAS BEEN RENDERED deficient
Similarity between transgenic and knockout animals
change is an inheritable trait and is passed onto successive generations
What are transgenic animals used for?
- models of disease to aid research (overexpression of tumour promoters)
- models to aid metabolism testing (overexpression of P450 enzyme)
- molecular pharming (produce human proteins and other pharmaceuticals in LARGE QUANTITIES) eg. pigs and cows that prdocue proteins that dissolve blood clots
What are knockout animals used for?
- models of disease to aid research (cancer, parkinson’s disease - knockout of genes)
- 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)
- assess tissue-specific pharmacology and physiology - receptors/channels
Conditional knockout animals are…
gene product knockout in a specific tissue
How to make transgenic mouse?
- pronucleus injection
2. blastocyst injection
What is pronucleus injection?
Injecting desired gene to pronucleus of fertilised mouse egg
DNA into pronucleus
What is blastocyst injection?
transforming embryonic stem cells with desired DNA then injecting it into blastocyst
Pronucleus injection METHODS
- Make transgene (coding sequence + promoter + enhancer sequence) by using recombinant DNA methods
- Incorporate transgene into a vector (or plasmid)
- Remove fertilised eggs from female mouse
- Inject transgene into MALE pronucleus
- Implant oocytes into foster female and develop normally
- Genotyping - do PCR analysis using tissue from the tail to determine if the offspring carries the transgene
- Establish a transgenic strain by mating the offsprings to make homozygous
(ones found in step 6 are heterozygous)
In pronucleus injection, why is transgene injected into male egg?
it is more successful
Survival rate of microinjection and implantation in pronucleus injection
very low, 10-30% survive the injection,
only 40% carry the transgene
gene and expression in pronucleus injection
we can see the gene everywhere in the body, but expression at specific parts
Advantage of pronucleus injection
applicable to most animals
all offspring will be heterozygous because we injected the transgene directly into the eggs
Disadvanage of pronucelus injection
DNA incorporates into the genome randomly (can knock out wrong cells), so it is important to screen
Low efficiency/success rate
What is a stem cell?
pluripotent cell capable of becoming any cell
Difference between blastocyst and blastocyte
Blastocyst is a hollow ball of cells made of outer trophoblast cells and inner cell mass
Blastocyte is just undifferentiated embryonic cell
When is ES cell harvested
from 3.5 day embryos
Blastocyst injection METHODS
- Make the transgene
- Isolate the ES cells and culture them on petri dish on a feeder layer of fibroblasts (so they proliferate and can be cultured continously)
- Expose ES cell to transgene by infection/transfection
- Select for successfully transformed cells on the dish (eg. antibiotic environment)
- Inject the ES cell to blastocyst of different characteristics mouse to make a blastocyst with both genes (original and new mouse)
- Reimplant it into the foster female and allow develop normally
- Select the chimera, establish the strain by mating 2 heterozygous to make homozygous (transgene in the germline)
What is germline?
A series of germ cells each descended or developed from earlier cells in the series
Why isn’t chimera or partially transgenic mice good enough?
because they are just mouses with heterozygous genes, we want them to have it homozygous
