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