Discuss animal and cellular models used to study disease mechanisms and therapeutic measures

1. Discuss animal and cellular models used to study disease mechanisms and therapeutic measures October 2007

2. Somedefinitions: • Gene transfer technology: DNA sequences produced in vitro are added to the genome (can be loss of function or gain of function). • Transgene: DNA tranferred into cell/animal model • Gene targeting: Use of gene transfer technology to modify the genome in a precise and predetermined way (mutations are designed then introduced into particular genes in vivo)

3. Some definitions (2): • Transgenic animal: An animal that contains the same additional DNA sequence in every cell (ie created by gene transfer) these include mice, drosophila, nematodes, fish, frogs, birds, rats, sheep… • Knock-in mutation A targeted mutation that replaces the activity of one gene by that of an introduced gene (usually an allele) • Knock-out mutation The targeted inactivation of a gene within an intact cell

4. Methods of gene transfer to cultured animal cells • There are four methods of gene transfer into cultured animal cells • 1. Transduction Uses viruses. The transgene is packaaged inside a virus particle, which uses its own way to insert the DNA into the host cell. 3 virus types i) High level transient transgene expression viruses, eg adenovirus, vaccinia, Semiliki ii) Long term stable transgene expression viruses, eg Epstein-Barr, herpes simplex, baculovirus iii) Stable transformation viruses, eg retroviruses, adeno-associated virus • 2. Transfection Chemical or physical means to persuade cells to take up transgenic DNA eg chemical tranfection (endocytosis), liposome-mediated tranfection (liposomes), lipofection (lipoplex), electroporation, receptor mediated endocytosis (cell surface ligands)

5. Methods of gene transfer to cultured animal cells (2) • 3. Direct transfer Physical introduction of DNA into the cell, eg microinjection, particle bombardment • 4. Bacterial gene Transfer Uses live invasive bacteria that undergo lysis within the anlimal cell, releasing transgenic DNA, eg, salmonella, shigella flexneri, Note: a selectable marker gene is used to isolate transformed cells after transformation. This is a gene which confers a property allowing transformed cells to grow and survive in the presence of an agent that kills or restricts growth of non-transformed cells.

6. Transgenic animals • To produce a transgenic animal stable gene transfer is needed, there are two ways to achieve this: 1. Directly. DNA is introduced selectively into germ cells, gametes or egg directly after fertilisation. If DNA integrates before the first zygotic division, all cells within the animal will contain the transgene 2. Indirectly. DNA is introduced non-selectively into the embryo before formation of its germline. DNA integrates randomly after the first zygotic division (embryo is mosaic). Embryos with the transgene in its germline will produce transgenic offspring.

7. Principles of direct gene transfer to animals(1)

8. Principles of direct gene transfer to animals (2) Germ cell transfer • Transgenic drosophila melanogastor (not mammals). • Uses transposable ‘P element’. Transgene is inserted inbetween 2 terminal sequences of the P element. DNA injected into germ-line precursor cells (Pole plasm of pole cells). • Also inject transposase enzyme (plasmid) to facilitate integration of new DNA into geneome. • Single copy of transgene is usually incorporated • Integration is random Sperm • Direct modification of gamete • 2 systems • Sperm mediated delivery. DNA is mixed with sperm in vitro, DNA binds to sperm heads. Followed by ICSI. Sperm DNA is not modified in this process. • Restriction enzyme mediated integration (REMI). Sperm nuclei are isolated, decondensed and mixed with plasmid DNA. Treated with RE to introduce nicks into DNA. Transplant into unfertilised egg where nicks are repaired and plasmid DNA is integrated into genome. Gamete genome is modified

9. Principles of direct gene transfer to animals (3) Egg/zygote • DNA injected into cytoplasm of fertilised egg. DNA randomly integrates into host genome. Usually a single copy of the DNA is integrated but can get upto 50. Immediate integration =>transgenic mouse • Can also inject DNA after 1 or 2 cell divisions and produce mosaic mouse. • Used in production of transgenic mice (40% success rate) also fish and amphibians Embryonic stem cells (ES cells) • Derived from 3.5-4.5 day old embryos (inner cell mass of blastocyst). • Cultured in vitro, (remain pluripotent). Inject back into host blastocyst and reimplant into pseudopregnant mother mouse. Developing embryo=chimera • ES cells can be maintianed indefinatley in culture (large scale experiments). • Also have high tendancy for homologous recombination which allows gene targeting.

10. Construction of a transgenic mouse

11. Principles of direct gene transfer to animals (4) Nuclear transfer • Replacement of an oocyte nucleus with the nucleus from a somatic cell. Somatic nucleus is then reprogrammed by the oocyte (Dolly, 1997) Retroviral transfer • Retroviral vectors transfer DNA into unselected cells of early embryos. Favoured method of producing mosaics animals. Can also have transfection of blastocysts and somatic cells…..

12. Control of transgene expression • To be of any use a transgene must be expressed when it is in a transgenic cell or animal • Transgenes are usually regulated by sequences present in the construct and can be expressed from constitutive promotors (eg viral promotors; SV40) or from cell/stage-specific promotors or inducible promotors (can be switched on/off in response to the presence of a particular chemical ligand). Inducible promotors give the most control over required gene expression • Position effects of transgenes can be avoided by making the transgene constructs large and including the gene’s regulatory elements eg YACs, transchromatin mice

13. Knock-out transfer A method of creating disease models to mimic human diseases (abolish/alter gene function) There are 3 ways this can be achieved • Gene targeting • Inhibition of gene expression • Insertional mutagenesis

14. Gene targeting Homologous recombination is used to introduce the transgene Allows creation of defined mutations at specific sites in the genome ( in vivo site directed mutagenesis) Requires a long sequence which is isogenic with the host genome Limited to mice and flies There are two gene targeting constructs: 1. Insertion vectors-Insertion of a vector by a single recombination event 2. Replacement vectors-Insertion of a portion of a sequence by a double reciprocal recombination or gene conversion event Used to create mouse models for: CFTR gene (cystic fibrosis), HBB/B-Globin (B-Thalassemia), GBA (Gaucher disease), SCA1/Ataxin Spinocerebellar ataxia type 1

15. Gene targeting by homologous recombination a) Insertion vector method b) Replacement Vector method

16. Inhibition of gene expression • Functional knock-out system (ie DNA sequence of target gene is not altered) • Used to generate phenocopies of a mutant phenotype • Can be used in cells and embryos • The transferred product inhibits expression of the host mRNA (RNA interferance) • Transferred products includes:antisense RNA, double-stranded RNA, small interfering RNA, ribozomes, antibodies, dominantly interfering proteins • Can introduce the inhibitors directly to give a transient effect (transfection/injection) or express them from a transgene (inverted repeat construct) to give a permanent effect • Can also block expression at the protein level eg by introducing DNA/RNA oligonucleotides to inhibit protein activity

17. a) stRNA pathway b) siRNA pathway

18. Insertional mutagenesis • Transgene randomly integrate into an existing gene thereby abolishing its function by introducing a mutation. Mutation is not defined or targeted to a specific site, ie process is random • Uses chemical mutagens and radiation (need to carry out positional cloning after to identify position of mutation) or gene transfer mutagenesis (uses sequence not found in host genome, gene trap. Can identify the host gene by its expression mirroring that of the transgene, giving functional information) • Used to study gene functions on a genome-wide scale (eg drosophila, mouse mutants) • Used to study X-linked DMD (mdx mouse), excessive neurofibromas (NF damselfish), missense mutations in canine factor IX gene (human homolog of haemophilia B), Diabetes mellitus (NOD mouse)

19. Creating disease models • Cell models can be used from human cell lines from affected individuals negating the need to produce artificial cell lines. Cellular models are often used first to identify potential chemicals as drugs, then animal models later for testing the drugs • Gain of function diseases which have a cellular phenotype can be modelled by transferring a disease-causing dominant gene into the appropriate cell type eg, 1. Xeroderma pigmentosa, Fanconi Anaemia (DNA repair) 2. Cancer (uncontrolled cell proliferation) • Loss of function diseases with a cellular phenotype can be modelled using eg RNA interference • Animal models provide a whole organism context and are therefore better for diseases without a cellular phenotype eg tremor

20. Animals used to study human diseases • Primates Theoretically should be the animal that mimics us most closely. Disadvantages are; long lives and less fecund than rodents therfore breeding experiments are long term, unpopular with the public • Mice Most widely used, small, cheap to maintain, short lifespan, prolific breeders, mapping of mutatnts is relatively easy with back-cross breeding and many polymorphic markers • Rats Used widely in physiological, pharmacological and behavioural studies because they are comparatively large. Some human disorders do not have good mouse models but have good rat models. Dense genetic maps are avilable for rats

21. Animals used to study human diseases (2) • Zebrafish Robust embryos that develop externally, therefore very useful for development studies. Short generation interval, Zebrafish genomes moderatley similar to human genome • Invertebrates and yeast Useful for drug testing systems as many genes and pathways are conserved between them and humans. Can be screened in large batches

22. Triplet animal models • Lin et al, Human Molecular Genetics, 2001, vol 10(2) created the CHL1 and CHL2 mouse lines by gene targeting of the Hdh locus (mouse equivalent to IT15) in ES with the selectable marker Hprt, followed by a second round of gene targeting to introduce an expansion. CHL1 mice have 50 CAG repeats and CHL2 mice have 80. Used to study neuronal dysfunction in HD. • Woodman et alBrain Research BulletinVolume 72, Issues 2-3, 30 April 2007, created the homozygous mouse model hdhQ150/Q150to study the effect of having two expanded HD alleles • Also mouse models for DRPLA (Atro-118Q) and SCA1, 3, etc

23. Wikipedia: • Animal model refers to a non-human animal with a disease or injury that is similar to a human condition. These test conditions are often termed as animal models of disease. The use of animal models allows researchers to investigate disease states in ways which would be inaccessible in a human patient, performing procedures on the non-human animal that imply a level of harm that would not be considered ethical to inflict on a human. • In order to serve as a useful model, a modeled disease must be similar in etiology (mechanism of cause) and function to the human equivalent. Animal models are used to learn more about a disease, its diagnosis and its treatment. For instance, behavioral analogues of anxiety or pain in laboratory animals can be used to screen and test new drugs for the treatment of these conditions in humans. • Animal models of disease can be spontaneous (naturally occurring in animals), or be induced by physical, chemical or biological means.

25. References: • Human Molecular Genetics, edition3, Strachan and Read • Lin et al, Human Molecular Genetics, 2001, vol 10 (2)