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Genetic manipulation of Animals How and
Why? Chris Tuggle Department of Animal
Science Iowa State University
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Overview A. Why transfer genes into animals?
B. Methods of gene transfer
C. Effects of gene transfer into animals
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Why transfer genes into animals?
Basic Research

• Model Biological processes

• Study gene regulation/function in context of
  animal.

• Identify and isolate new genes of interest

• Mark cell lineages for developmental studies

Several advantages of Transgenesis over other
methods to study Biology
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Why transfer genes into animals?
Applied Research

• Produce therapeutic proteins

• Cure disease correct of genetic defects.

• Generate models for human disease.

• Production of xenotransplant donors.

• Production of superior animals.

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Why transfer genes into animals?i.e., Why not
do this research in other ways?

• 1. Process under study not applicable to culture
  systems.

• Cell type to be studied cannot be cultured.

• Inability to replicate in vivo environment in
• tissue culture (celllt-gtcell tissue
  interactions).

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Why transfer genes into animals?
2. Developmental studies require a living mammal
to study certain aspects of

• Implantation events/physiology

• Maternalfetal interactions

• Birth events/physiology

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Why transfer genes into animals?

• 3. Animal tissue expression required for protein
  effectiveness or activity

• Mammal-specific modifications to protein for
  activity.

4. Protein production in transgenic animal
cost-effective
5. Scientific goal requires use of animal

• Human gene therapy, xenotransplantation
• Animal genetic improvement

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Sidelight The Mouse as the model research mammal

• Advantages for the Use of Mice in Genetic
  Manipulation Research
• Many genetic manipulations possible (add genes,
  delete genes)
• Fast reproductive cycle for a mammal -egg to egg
  about 3.5 months
• Many genetic strains and mutants described
• Best characterized animal which is closest to
  humans of laboratory animals, most directly
  useful for biomedical genetics research

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Sidelight The Mouse as the model research mammal

• Disadvantages for the Use of Mice in Genetic
  Research
• Large genome (can be difficult to screen for
  certain manipulations)
• Long gestation time (relative to invertebrates
  flies, worms)
• Inaccessibility of the fetus for
  observation/manipulation especially relative
  to vertebrate species like chicken and zebrafish
• Expense (complicated growth media uterus)
• Ethical concerns require good justification

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Categories of Genetic Manipulation

• Germ-line Genetic Modification

• Genetic modification is performed early enough in
  developmental life of the animal that all cells
  of resulting transgenic animal carry the
  modification.

• Thus germ cells (sperm or egg) are modified and
  will transmit the transgene to the next
  generation as any other chromosomal gene

• Examples

• Expressing foreign proteins in milk of transgenic
  mice

• Modifying pigs to serve as xenotransplantation
  donors
• Creating livestock with superior production traits

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Categories of Genetic Manipulation
Somatic Cell Genetic Modification

• Modification is performed relatively late in the
  life of the animal (often in the young adult) so
  that one or only a few cell types are genetically
  changed.

• Thus as germ cells are not modified, the
  transgene which may exist elsewhere in the animal
  cannot be passed to the next generation.

• Examples

• Human ex vivo gene therapy

• Injection of DNA into mammary tissue
• to identify optimal transgene construct

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Types/Uses of Transgenes Constructs

• 1. Reporter Gene Fusions

• Used to dissect gene expression mechanisms
• -to find DNA elements controlling expression

• Reporter is an easy-to-analyze protein usually
  an enzyme that produces a colored or fluorescent
  product.

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Gene Regulation
Without Enhancer
With Enhancer
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Types/Uses of Transgenes Constructs

• 2. Knockout/Knock-in Constructs

• For targeted disruption or specific modification
  of a gene using homologous recombination.
• To study the function of the gene.
• KO to determine general function
• KI to precisely modify genetics.

• Selectable marker gene is inserted into a gene in
  cultured cells, then these cells are used to
  create a mouse with the disruption in every cell.

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Positive-Negative Selection- Technology to Enrich
for True Homologous Recombinants
Negative TK activity can be selected against
Generalized contruct for HR
Positive- select for Neomycin resistance
Neor cassette
Exon disrupted
Thymidine kinase gene
X
X
Genomic sequences available for homologous
recombination
Gene function disrupted at this allele
Neor cassette
Exon disrupted
Exon disrupted
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Types/Uses of Transgenes Constructs

• 3. Directed RNA/Protein Expression

• For specific production of a specific gene
  product such as a pharmaceutical protein which is
  missing in a patient with inborn error of
  metabolism.

• Many levels of control may have to understood for
  accurate direction of expression
• Tissue specificity
• Time or stage specificity
• Subcellular localization signals
• Excretion signals

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Methods of Gene Transfer
1. Pronuclear microinjection of fertilized
embryos

• Many papers describing successful use

• Production of multi-subunit enzyme activity by
  multiple gene transfer Prunkard (1996)

2. Recombinant retroviral infection

• May be used more in the future with high
  resolution ultrasound injection in utero

3. Injection of blastocysts with manipulated
embryonic stem cells for knockout/knock-in mice
production

• Homologous recombination, many papers

4. Transfer of in vitro modified nuclei into
enucleated embryos

• Again homologous recombination
• Few papers in field- mouse, pigs (sheep?)
• KO work published only

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Methods of Gene Transfer
1. Pronuclear microinjection of fertilized
embryos

• Many papers describing successful use

• Production of multi-subunit enzyme activity by
  multiple gene transfer Prunkard (1996)

2. Recombinant retroviral infection

• May be used more in the future with high
  resolution ultrasound injection in utero

3. Injection of blastocysts with manipulated
embryonic stem cells for knock-out/knock-in
mice production

• Homologous recombination, many papers

4. Transfer of in vitro modified nuclei into
enucleated embryos

• Again homologous recombination
• Few papers in field- mouse, pigs (sheep?)
• KO work published only

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Methods of Gene Transfer
1. Pronuclear microinjection of fertilized
embryos

• Many papers describing successful use

• Production of multi-subunit enzyme activity by
  multiple gene transfer Prunkard (1996)

2. Recombinant retroviral infection

• May be used more in the future with high
  resolution ultrasound injection in utero

3. Injection of blastocysts with manipulated
embryonic stem cells for knock-out/knock-in
mice production

• Homologous recombination, many papers

4. Transfer of in vitro modified nuclei into
enucleated embryos

• Again homologous recombination
• Few papers in field- mouse, pigs (sheep?)
• KO work published only

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Generation of Specific Mutation In Living Mice
How does one screen for a targeted cell?
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Methods of Gene Transfer
1. Pronuclear microinjection of fertilized
embryos

• Many papers describing successful use

• Production of multi-subunit enzyme activity by
  multiple gene transfer Prunkard (1996)

2. Recombinant retroviral infection

• May be used more in the future with high
  resolution ultrasound injection in utero

3. Injection of blastocysts with manipulated
embryonic stem cells for knock-out/knock-in
mice production

• Homologous recombination, many papers

4. Transfer of in vitro modified nuclei into
enucleated embryos

• Again homologous recombination
• Few papers in field- mouse, pigs (sheep?)
• KO work published only

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Steps in Nuclear Transfer and Cloning
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Precise Genetic Modification NT of Gene Targeted
Nuclei
Embryonic Stem cells
Fetal Fibroblasts
Gene Targeting by Homologous Recombination
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Effects of Gene Transfer into Animal/Humans

• 1. Disease Models/Curing Disease
• 2. Studying Gene Regulation in the Whole Animal
• 3. Mutagenesis - Study Gene Function
• 4. Gene Product Synthesis/Breed Improvement
• 5. Human Gene Therapy

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Effects of Gene Transfer into Animal/Humans

• 1. Disease Models/Curing Disease

• Dominant disease models
• Oncogenesis - breast cancer mouse model (mice
  with strong promoter expressing oncogene are
  cancer-prone)
• Spongiform encephalopathy (mad cow disease) is
  caused by prions. Transgenice mice expressing
  hamster prion gene have scrapie lesions and
  transgenic mice are more easily infected by
  scrapie agent.

• Human immune system model for AIDS research
• Replacement of parts of mouse immune system with
  that unique to humans.

• Curing inborn errors of metabolism
• Usually recessive mutations, so only need to ADD
  gene information- many examples.

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• 2. Studying Gene Regulation in the Whole Animal
• Main method is to use reporter genes to identify
  regulatory regions that express reporter in
  specific cell types.

• Tissue-specific--mammary gland, liver, muscle.

• Region-specific--developmentally regulated genes.

• Stage-specific-example globin gene family.

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In vivo testing of DNA regulatory sequences
Wild Type Mutant Flanking Sequence Flanking
Sequence
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• 3. Mutagenesis - Study Gene Function

• Dominant mutations

• Excess human Growth Hormone - giant mouse

• Interfering with formation of multiprotein
  structures and assemblies

• Recessive mutations

• Null mutations (targeted mutations using ES
  cells) many examples now

• Examples
• transgenic mice with knockout in tumor suppressor
  gene (p53) are tumor-prone.
• NRAMP genetic proof

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• 4. Gene Product Synthesis/Breed Improvement
• History of Transgenic Farm Animals
• 1985-1992
• Modification of animal for production trait
  improvement
• GH or IGF overexpressing transgenes
• 1988-present
• Modification of animal to produce
  pharmaceuticals-
• Gene Pharming
• Blood clotting factors
• Nutrition additives
• Other metabolism enzymes

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• 4. Gene Product Synthesis/Breed Improvement
• 1985-present
• Modification of animal to serve as donor for
  human transplantation
• Xenotransplantation major problem that needs
  to be overcome is hyperacute rejection
• Transgenic organ transfer not performed in humans
  yet.
• 1997-2000
• Use of nuclear transfer to clone animals adult,
  fetal cells as nuclei donor. Use of genetically
  manipulated fibroblast cells and subsequent
  nuclear transfer to generate transgenic sheep,
  cattle, mice (gene addition and locus-specific
  studies(sheep and mice)).
• 2001-2
• Cell culture approaches to modify developing
  chicken cells. (2001)
• Use of nuclear transfer to produce targeted
  modifications in pigs (2002)- first knock-out in
  pigs

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Uses for Gene Trap technology in Functional
Genomics

• Gene Trap- a insertional mutagenesis technique
  where the mutatgen is assayable marker gene
  (lacZ)
• Used to identify and study genes involved in
  development because different blue stain patterns
  can be easily screened.
• Used extensively now for functional genomics
• a) generate mutations in many new genes
• b) see expression pattern in single copy also
• c) if breed to homozygosity, observe
• phenotype of insertional mutation at this
  gene

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Use transgenesis and Gene Trap construct to
find new genes
Gene Trap - promoterless lacZ gene - no
activity unless integrates into gene - blue
stain shows where new gene is normally
expressed
LacZ
Gene B-like Expression possible
No Expression
Gene A
Gene B
Gene C
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Gene trap transgenic embryos
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Conclusions

• Many useful methods to modify the genome of mice
  to understand and model biology

• Recent developments in cloning and nuclear
  transfer holds promise in gene transfer into
  large animals for genetic improvement

• New uses for transgenic mice in the Human
  Genome Project

• All such manipulations should be ethically
  justified

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DNA Microinjection of Fertilized Oocyte