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Define transgenic and cloning
- Transgenic = deliberate modification of the genome of an animal
- Cloning = transfer of entire genome from one individual to create another
What is the order of components in a transgene?
Promoter sequence, gene, poly A sequence
What is a promoter sequence?
The promoter may be used to direct expression in all cells of the transgenic animal or used to direct expressional to a particular tissue e.g. for expression of proteins in milk the casein promoter is used
What is a poly A sequence?
They are required to guarantee efficient processing of the mRNA (gene)
Describe the steps involved in pronuclear DNA microinjection
- Eggs harvested and fertilised in vitro
- Fine needle injects transgene containing solution (50-500 copies)
- Several embryos are implanted into a pseudopregnant foster mother
- DNA from tail samples is multiplied by PCR and analysed for presences of 'new' DNA
- Mice showing optimum expression of the 'new' gene are selected. The 'new' DNA will be stable in further generations.
What are the advantages of pronuclear DNA microinjection?
- Simple technique
- Direct injection of DNA solution into fertilised embryos
- Capable of germ line transmission if integrated into reproductive stem cells
- Large DNA constructs
What are the disadvantages of pronuclear DNA microinjection?
- Random integration at low efficiency
- More than one copy integrates
- Only 10-40% of viable offspring are transgenic
- Chromosomal site integration can result in effects on transgene (and native gene) expression
What can happen if transgene is inserted into embryos past the 1 cell stage?
There is the possibility of the animal begin a mosaic
Describe the steps involved in embryo cell transfer?
- Embryonic stem cells are grown in culture. They can be modified in culture.
- Stem cells are injected into blastocyst.
- Altered blastocysts are implanted into surrogate mother.
- Offspring are mosaics.
- Further breeding is required to get homozygous expression in offspring.
What are the advantages of embryonic cell transfer?
- Targeting to specific insertion site is possible
- Can check location/expression of transgene in vitro
- Can be used to derive knockout animals
- Can be stored indefinitely
What is a disadvantage of stem cell transfer?
Embryos are at the multinuclear stage so result in mosaics - require further breeding
Describe the steps involved with retroviral vectors in transgenics
- Transgene constructs are inserted into retroviral particles used to infect embryos
- Animals produced are mosaics
- Further breeding required to get homozygous expression in offspring
List three pros of transgenics
- Specificity - the characteristic required can be targeted
- Speed - characteristic potentially acquired in few generations
- Flexibility - cross species possible e.g. mouse with human genes
List two cons of transgenics
- Animal health - insertion of transgene may upset genome expression
- Dissemination - escaped transgenic animal may pass transgene into the wild
List examples of uses of transgenics
- Molecular 'pharming'
- Improved production
- Environmental protection
- Targeting disease vectors
What are some applications of cloning?
- Perpetuation of transgenic animals
- Retention of 'unique' characteristics/abilities
- Endangered species
- Harvesting of tissues/embryonic stem cells
Describe the steps involved in cloning
- Nucleus/DNA is taken from an adult somatic cells. DNA is removed from an egg cell.
- Somatic cell nucleus is inserted into enucleated egg cell.
- Egg cell is stimulated with a shock and starts to divide
- In culture this single cell forms a blastocyst with identical DNA to the donor somatic cell
- Embryo implanted into surrogate mother
- Clone is born
What are some concerns with cloning?
- Many attempts before success
- Short life span of clones
- Clone may differ from parent due to epigenetics, environmental factors, x-chromosome inactivation
What is stem cell therapy?
The use of stem cells to alleviate disease in animals and humans
Why are embryonic stem cells great for therapy?
As they are pluripotent
What is the main issue with using embryonic stem cells?
Moral and ethical concerns
What other types of stem cells can be used instead of embryonic stem cells?
- Adult stem cells
- Induced pluripotent stem cells
What are stem cells used to treat in veterinary medicine?
Dogs and cats - arthritis. Horses - arthritis and tendon injury.