Transcript Document

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 allpurpose stem cell.
Organisms utilized as transgenic
models
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Arabadopsis (plant)
C. elegans (worm)
Fruit flies
Xenopus (frog)
Zebrafish
Mice
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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.
B.
C.
Physiologically similar to humans.
Large genetic reservoir of potential models has been
generated through identification of >1000 spontaneous,
radiation- or chemically-induced mutant loci.
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.
E.
F.
>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.
Allow study of disease on uniform genetic background.
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
Brown (or mixed)
coat color mice are
chimeras
brown
black
Look for chimeric progeny
(coat color)
Breed for germline transmission
brown
black
Check offspring for coat color
brown
Isolation of ES cells
Gene targeting method to produce null mutant
(“knockout”) or mutant (“knock-in”) mice
Homologous recombination
neo
HSV tk
neo
G418R GANCR
Random integration
neo
HSV tk
neo
G418R GANCS
HSV tk
Generation of mutant (chimeric) mouse strain
+/+ ES cells from brown mouse
electroporate targeting vector
homologous recombination
+/- ES cells
microinjection into black embryo
X
chimera
screen chimera progeny
+/+
+/+
+/-
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
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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.