Transgenics, Cloning, Stem cells and Potential Applications

What is molecular cloning?

-manipulation of nucleic acid
-cloning, restriction digestion, mutagenesis, gene expression
-carried in bacteria elements allow amplification/expression of changed DNA
-develop transgenic animals, recombinant vaccines

What is the definition of transgenic?

deliberate modification of genome

What is the definition of cloned?

transfer of entire genome from one individual to create another

What are transgenic animals?

-genetically altered animals w/additional DNA sequences
-particular sequence deleted from/inactivated in genome
-breeding lead to lines w/genetic alteration in cells

How are gransgenic large animals made?

-added transgene construct
-contain promotor, gene, poly A sequences
-gene expressed is cloned downstream of promoters
-promoter use direct expression in cells of transgenic animal or use direct expression to particular tissue, tissue specific expression
-poly A sequences required to process mRNA

What are the techincal approaches to make GM animals?

-pronuclear DNA microinjection
-gene targeting of embryonic stem cells
-retroviral vectors
-gene editing: molecular scissors

What occurs during pronuclear microinjection?

1. eggs harvested after super ovulation

2. newly fertilised egg in vitro

3. needle injects transgene solution w/50 to 500 copies

4. embryo implanted in pseudopregnant foster mother

5. birth litter

6. DNA from tissue samples is multiplied by PCR and analysed for new DNA

7. Optimum expression of new gene are selected, new DNA stable in further generation

What are the advantages and disadvantages of pronuclear DNA microinjection?

Advantages:
-simple technique
-direct inj DNA solution into fertilized embryos
-embryo at 1-cell stage, integration after 1 to 2 cell divisions=mosaic
-capable of germline transmission
-large DNA constructs

Disadvantages:
-random integration at low efficiency
-more than one copy integrates, copy dependent transgene expression
-10 to 40% viable offspring are transgenic
-chromosomal site integration result in effects on transgene expression

What occurs during embryonic cell transfer?

-embryonic stem cells grow in culture
-pluripotent and differentiate into any cell type
-modified in culture and injected into blastocyst
-blastocysts are implanted into surrogate mother
-offspring are mosaics

What are the advantages and disadvantages of embryonic cell transfer?

Advantages:
-targeting specific insertion site possible
-check location/expression transgene in vitro
-used to derive knockout animals
-stored indefinitely

Disadvantages:
-embryos multicell stage

What occurs during retroviral vectors?

-transgene construct packaged in retroviral particles used to infect embryos
-efficient transfer of single copy of transgene at single chromosomal site
-carried out at multicellular stage=mosaic
-animals bred for many generatios generate transgenic animal homozygous for transgene in all cells

What is the rate of transgenics?

-slow success rate
-become estabilshed in germline to be passed on
-require breeding from F1 transgenics to develop homozygous animals

What are the pros and cons of transgenics?

Pros:
-specificity: characteristic required is targeted
-speed: charateristic potential acquired in few generations
-flexibility: cross species possible

Cons:
-Animal Health: insertino transgene upset genome expression
-potential risk to environment

What are categories of transgenic animals?

-study of cellular processes
-producers of human therapeutics: molecular pharming, xenotransplantation
-enhanced food quality or efficiency of production
-improve animal health through development of resistant animals or understanding disease process
-producers of industrial products
-modifications address environmental concerns

What are features of molecular pharming?

-transgenic livestock: transgene is directed for expression in milk
-milk harvested from milk and transgenic protein purified
-purified protein administered to target species

What are features of cenotransplantation?

-transplantation of organ, tissue, cells from one species to another
-safe product, good availability, lower health care costs, better scheduling

Why is an animal cloned?

-no guarantee offspring will have good expression of transgene
-genetically identical offspring
-cloning captures traits might be multigenic and genetics unknown
-can capture genetic material of sterile/neutered animals
-conservation
-harvesting ot tissues/embryonic stem cells

How was Dolly created?

-1st animal cloned from adult cell

1. nucleus/DNA taken from donor adult somatic cell

2. DNA removed from egg cell of another animal

3. Somatic cell nucleus inserted into enucleated egg cell

4. newly formed egg stimulated with shock and starts to divide

5. culture single cell forms blastocyst w/identical DNA to donor somatic cell

What are the concerns of cloning?

-1 out of 277 attempts succeeded making Dolly
-efficiency <5%
-short lifespan of clones
-epigenetics, x-chromosome inactivation, environmental factors
-ethics

What is stem cell therapy?

-use stem cells to alleviate disease in animals and humans
-bone marrow therapy for anemia, lymphoma, leukemia

What are features of embryonic stem cells?

-pluripotent potential
-divide w/limitless potential
-grow in undifferentiated states in culture
-contribute to all somatic tissues and germ cells reintroduced into embryo

What are issues with therapeutic cloning?

-human somatic cell nuclear transfer requires human eggs
-potential creating human reproductive clone if blastocyst implanted
-cost and inefficiency
-transmission of animal viruses: stem cells cultured in products contain animal products and successful culture on murine feeder layers
-moral and ethical concerns outweight all other considerations

What are features of induced pluripotent stem cells?

-derived from non-pluripotent cells, adult comatic cell
-inducing expression of certain fenes

Technical hurdles
-low efficiency
-potentiwal risk of viral vectors