Animal transgenesis Flashcards
What is transgenesis?
Cross species transfer of genetic material
What makes an animal transgenic? (2)
A genetically modified animal containing a gene from another species. It can be gene addition/deletion or gene modification.
What is the point in gene knockout?
To determine the function of specific genes
What are the objectives of genetic modification in animal transgenesis?
To produce a stable and heritable change in the genetic makeup of an animal which doesn’t occur under standard conditions. Add/remove genes. Place genes at specifc loci (knock-in). Can also alter the gene expression levels.
What are three reasons for modifying animals?
- Gene function / model disease
- Modify production traits
- Organs for xenotransplantation
How can disease resistance be a result of modifying animals?
Removal of receptors which make the animal susceptible to disease
What is an example of neutraceuticals being produced via modifying animals?
Modification of fatty acids present in meat
What are the four methods to produce transgenic animals?
- Pronuclear injection
- Embryonic Stem Cells
- Somatic Cell Nuclear Transfer
- RNA guided engineered nucleases
What is pronuclear injection?
The injection of linearised DNA (gene of interest) (several hundred copies) into either one or both of the pronuclei of the fertilised zygote
Why is the DNA linearised and why so many copies?
Multiple copies at random insertion sites, enhancing chances of transgene insertion. Linearised is easier to integrate than circularised, as well as having multiple ends making integration into the genome easier.
Which type of cell and why does the transgene need to be inserted into?
Germ cells because these give rise to the founding embryo
What two important outcomes come from screening many animals for the transgene?
Determines which animal(s) have the transgene present and in WHICH tissue
Following injection, embryos are returned to the surrogate recipient. Mice = 10-20% transgenic offspring, farm animals is 1-10%
What are four limitations of microinjection technology?
- Most animals are not transgenic
- Variation in transgene expression from founder to founder
- Mosaicism
- Flock/herd development is slow
What is meant by mosaicism?
Segregation of DNA into different cells, with different expression levels, locations. Hard to determine the impact of the transgene due to complex phenotypes.
What are three applications for recombination in mouse ES Cells?
- Mouse models of human diseases
- Gene function studies
- Immunology
For gene targeting in mouse ES Cells, what are the first three steps?
- Design a targeting vector with an antibiotic resistance gene with flanking regions homologous to the gene of interest.
- ES Cells are isolated from mice embryos/blastocysts and the vector is introduced via electroporation.
- The wild-type gene already present is replaced by the modified gene (in vector) via homologous recombination.
Steps 4-6
- Antibiotic selection is applied to the ES Cells - only the cells which undergone homologous recombination will survive.
- Screen clones which were successful through PCR.
- Inject ES Cells into blastocysts, then implant into surrogate mouse.
What type of mouse is produced and why?
Chimeric mouse because it has modified and unmodified cells
Step 7
Breed chimeric mice with normal mice to produce offspring with the modified gene.
Step 8
Breed the mice continuously until homozygosity is achieved (two copies of the modified gene).
What is Somatic Cell Nuclear Transfer (SCNT)?
The nucleus of a somatic cell is transferred into an enucleated oocyte. The resulting cell is stimulated to divide and develop into an organism genetically identical to the donor of the somatic cell.
What are four benefits of SCNT?
- Genetic modification
- Cloning from adult animals
- Rapidly produce an unlimited source of donor cells
- All animals are transgenic
100% success in every cell containing the transgene, but 5% are viable to survive. Microinjection have the majority of embryos surviving but few contain the transgene.
What are the four main transcription factors responsible for reprogramming of a somatic cell back to a pluripotent state (induced pluripotent stem cell)
IPSC conversion require Oct4, Sox2, Klf4, and c-Myc
What are the first two steps in IPSC & SCNT?
- Adult cells are reprogrammed to a pluripotent state
- At the same time, an early stage developing embryo is obtained, consisting of only a few cells
3 & 4
- IPSC are introduced into the embryo by injection or incorporation during its culture
- IPS Cells integrate into the developing tissues and contribute to various tissues and organs
What is created now?
Chimeric organism
The organism now has cells derived from both IPSC and the host embryo. IPS is not commercial yet. NT has a 5% success rate and microinjection is 10-20% in mice.
Why is xenotransplantation using pigs useful?
Similar organ size and it only takes 6 months to produce a pig
What are transgenic animals used for? (3)
- To alter specific traits
- The production of specific products for pharmalogical applications
- Used as models for studying diseases and gene function
How are transgenic animals used to study diseases?
They can be engineered to carry specific mutations associated with human disease. Study overexpression or knock out of genes. Can test safety and efficacy of therapeutic drugs.
