18. Transgenic Models

Approaches Used in the Analysis of Mammalian Development Observations during embryogenesis Phenotypic analysis of developmental mutants Cloning and analysis of expression of developmental genes Generation of mouse mutants by gene targeting Gene transfer in established cell lines and transgenic mice Nuclear transfer (“cloning”)

Transgenesis – definitions Transgenic animal – one that carries a foreign gene that has been deliberately inserted into its genome. Chimeric animal – one that carries an altered gene introduced using manipulated embryonic stem (ES) cells. Some tissues are derived from cells of the recipient blastocyst; other tissues are derived from the injected ES cells. Knockout mutation – replacement of a gene segment by homologous recombination that normally results in a nonfunctional or “null” allele.

Transgenesis – Definitions (continued) Knock-in mutation – similar to a knockout mutation, except mutation is usually a point mutation that results in a partially functional or nonfunctional allele. Nuclear transfer – technique used to create a “clone.” The nucleus from an adult somatic (differentiated) cell is inserted in the emptied cytoplasm of an egg cell. The egg cell reprograms the adult cell’s genes so that it behaves like an all- purpose stem cell.

Organisms utilized as transgenic models Arabadopsis (plant) C. elegans (worm) Fruit flies Xenopus (frog) Zebrafish Mice Rats Pigs Sheep Goats Cows

Types of transgenes Small recombinant DNA molecules – genes or cDNAs linked to DNA sequences that allow correct expression by the cells of the host Reporter constructs – desired gene promoter linked to expression cassette that can be assayed; e.g., GFP, lacZ, luciferase Large native DNA molecules – yeast artificial chromosomes (YACs) or bacterial artificial chromosomes (BACs)

1.Frogs were among the first transgenic animals produced. 2.Isolated DNA was not transferred, nuclei were utilized (setting the stage for recent cloning technology). 3.Microinjection methodology was established.

Mouse Models of Human Disease: Utility A.Physiologically similar to humans. B.Large genetic reservoir of potential models has been generated through identification of >1000 spontaneous, radiation- or chemically-induced mutant loci. C.Recent technological advances have dramatically increased our ability to create mouse models of human disease. 1. Development of high resolution genetic and physical linkage maps of the mouse genome – facilitates identification and cloning of mouse disease loci. 2. Transgenic technologies that allow one to ectopically express or make germline mutations in virtually any gene in the mouse genome; i.e., transgenic mice, ES cell knockouts. 3. Methods for analyzing complex genetic diseases.

Mouse Models of Human Disease: Utility (Continued) D.>100 mouse models of human disease where the homologous gene has been shown to be mutated in both human and mouse. 1. Mouse mutant phenotype very closely resembles the human disease phenotypes. 2. Provide valuable resources to understand how the diseases develop and test ways to prevent or treat these diseases. E.Allow study of disease on uniform genetic background. F.Will aid in identifying modifier genes and are well poised to lead us into the new era of polygenic disease research.

Production of transgenic mice (overview)

Production of transgenic mice

Example of transgenic mouse

Production of chimeric mice (overview) Inject genetically modified embryonic stem (ES) cells from brown mouse into black mouse blastocyst (embryo) Implant into foster mother Look for chimeric progeny (coat color) Breed for germline transmission Check offspring for coat color brown black brown Brown (or mixed) coat color mice are chimeras

Isolation of ES cells

Gene targeting method to produce null mutant (“knockout”) or mutant (“knock-in”) mice

Homologous recombination neo HSV tk neo G418 R GANC R

Random integration neo HSV tk neo G418 R GANC S neo HSV tk neo

Generation of mutant (chimeric) mouse strain electroporate targeting vector microinjection into black embryo X +/+ ES cells from brown mouse homologous recombination +/- ES cells screen chimera progeny +/+ +/- chimera

Chimeras Non-chimeras are black.

Production of chimeric mice

Knockout mice Valuable for discovering function(s) of genes for which mutant strains were not previously available. Generalizations: 1. Mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. 2. Most genes are pleiotropic; that is, they are expressed in different tissues in different ways and at different times in development.

Studies Using Transgenics Rescue of mutants Mice as bioreactors – synthesis of human monoclonal antibodies. Structure-function relationships Mouse models of human disease – analysis of cellular and molecular mechanisms underlying pathogenesis and for testing drug regimes and/or gene therapies.

Mouse models listed by primary organ or tissue system affected Disorders of neural crest derivatives – 11 Disorders of vision and hearing – 7 Disorders of bone, skin and connective tissue – 16 Neurological and neuromuscular disorders – 22 Neoplastic disorders – 11 Immunological and hematological diseases – 17 Metabolic and hormonal diseases – 24 Human diseases with polygenic etiology – 6

Examples of mouse models of genetic diseases Sickle cell disease – combination of knockout and transgenic technologies. Endogenous globin genes made null, human globin genes introduced as transgenes. Cystic fibrosis – mainly knockout models, some transgenic. Polycystic kidney disease – knockout models only. Narcolepsy – knockout model.

Narcoleptic mouse, a.k.a. model of medical students attending early morning lecture.