Transgenic animals

Transgenic animals

• For what reason?

• How to do it...
• Can you clone an animal?
• How can you use it?
 Transgenic animals
Why?
• Selective breeding is performed since
centuries.
• Breeding is time consuming.
• Crossing in properties is time consuming.
• Only a limited number of properties
available.
 Transgenic animals
For what reason?
• 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
Transgenesis in Mice
Retroviral Vector
 Transgenesis in Mice
Retroviral 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 Mice
Microinjection
Transgenesis in Mice
Microinjection
 Transgenese in Mäusen
Mikroinjektion

• Low efficient mechanism of transgene

integration (5-10%)
• Transfer of large genes possible
• random insertion of the transgene
• Variable expression levels
no selection of the transgene necessary
Efficiency of Microinjection
 Transgenesis in Mice

GFP under the CMV Promoter

Transgenesis in Mice
Stem cell Method
Embryonic Stem cells
~30 cells 200-250
cells
Embryonic Stem cells
Stem cell differentiation
Gene Knockout

Chromosome

Target Vector

Chromosome
Negative Selection
Positive Selection
Vasectomia of male mice
Blastocysts
Injection of embryonic stem cells
Implantation of Blastocysts
Implantation of Blastocysts

3
Chimäre Mäuse
Black mouse -
no ES cell integration

Chimeric mouse -
high ES cell integration

Chimeric mouse -
low ES cell integration
Cross breeding strategies
 Transgenesis in Mice
Stem cell Method

• Efficient transfection
• positive-negative Selection
• Targeted insertion of the gene
• Characterization before animal
generation
• Time and cost intensive
 Transgenesis in Mice
“Smart“ recombination Methods
FRT FRT
1 βgalneo 2 3
loxP loxP

lacZ-tagged insertion allele

Flp recombinase Cre recombinase

FRT FRT

1 2 3 1 βgal
βgal
neo 3
loxP

pre-conditional allele (wild-type) lacZ-tagged null allele (Δ exon)

Cre recombinase

1 3

null allele (Δ exon, frameshift, NMD)

 “Knock-in” mouse model for
nephrogenic Diabetes insipidus
E242X loxP loxP

Exon 1 Intron 1 Exon 2 NeoR Exon 3/3’-UTR

K.O. Wildtype

K.O.
 Pathomechanism of
nephrogenic Diabetes insipidus
Mouse Model of the X-linked NDI
 Transgenesis in mice
Use

ß-galactosidase under the Neurogenin promoter

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: Cryo
conservation of cells
costs: $895 - $1395
• Texas A&M takes
$250,000 for cloning
a cat
Identification of transgenic Mice