talk transgenic technologies · transgenic animals for what reason? transgenesis in mice retroviral...
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Transgenic animals
Transgenic animals
• For what reason?• How to do it...• Can you clone an animal?• How can you use it?
Transgenic animalsWhy?
• Selective breeding is performed since centuries.
• Breeding is time consuming.• Crossing in properties is time consuming.• Only a limited number of properties
available.
• Introduction of a desired property(hypothesis driven; without hypothesis)
• Fast generation of animal lines carrying the desired property
• Animal model for human diseases• Animal system to produce biomolecules
(„Pharming“)• Xeno-Transplantates
Transgenic animalsFor what reason?
Transgenesis in MiceRetroviral Vector
• Efficient mechanism of transgene integration • Transfer of genes < 8 kb possible• random insertion of the transgene• Retroviral contamination of the transgenic
animal
Transgenesis in MiceRetroviral Vector
Transgenesis in MiceMicroinjection
Transgenesis in MiceMicroinjection
Transgenese in MäusenMikroinjektion
• Low efficient mechanism of transgeneintegration (5-10%)
• Transfer of large genes possible• random insertion of the transgene• Variable expression levels
no selection of the transgene necessary
Efficiency of Microinjection
GFP under the CMV Promoter
Transgenesis in Mice
Transgenesis in MiceStem cell Method
Embryonic Stem cells~30 cells 200-250
cells
Embryonic Stem cells
Stem cell differentiation
Gene Knockout
Target Vector
Chromosome
Chromosome
Negative Selection
Positive Selection
Vasectomia of male mice
Blastocysts
Injection of embryonic stem cells
Implantation of Blastocysts
3
Implantation of Blastocysts
Black mouse -no ES cell integration
Chimeric mouse -high ES cell integration
Chimäre Mäuse
Chimeric mouse -low ES cell integration
Cross breeding strategies
• Efficient transfection• positive-negative Selection • Targeted insertion of the gene• Characterization before animal
generation• Time and cost intensive
Transgenesis in MiceStem cell Method
1 2 3
lacZ-tagged insertion allele
FRTFRT
loxPloxPβgalneo
lacZ-tagged null allele (Δ exon)
1 βgal 3FRTFRT
loxP
Cre recombinase
βgalneo
pre-conditional allele (wild-type)
1 2 3
null allele (Δ exon, frameshift, NMD)
Flp recombinase
1 3
Cre recombinase
Transgenesis in Mice“Smart“ recombination Methods
“Knock-in” mouse model fornephrogenic Diabetes insipidus
loxP loxPE242X
K.O.
WildtypeK.O.
Exon 1 Intron 1 Exon 2 NeoR Exon 3/3’-UTR
Pathomechanism of nephrogenic Diabetes insipidus
Mouse Model of the X-linked NDI
ß-galactosidase under the Neurogenin promoter
Transgenesis in miceUse
Cloning by nucleus transfer
„Dolly“• 1997, first living
offspring derived from a differentiated cell
• Is Dolly a real clone?
„Gene“
• 1997, first living bovine offspring derived from a stem cell of a 30 days-old fetus
• Difference compared to Dolly?
„Cc:“• First clone cat
• Derived from fibroblast cell by nucleus transfer.
• Coat color differs between the genetic mother and the offspring
Commercialized pet cloning
• available: Cryoconservation of cellscosts: $895 - $1395
• Texas A&M takes $250,000 for cloning a cat
Identification of transgenic Mice