Anja Scheller Transgene Mäuse in der Forschung –

Anja Scheller
Transgene Mäuse in der Forschung –
von der Erzeugung bis zur Anwendung
Department of Molecular Physiology
Institute of Physiology - Medicine School
University of Saarland/Homburg
• Erbsubstanz à DNA (desoxyribonucleic acid)
• helixförmiger Doppelstrang aus den Grundbausteinen A,
G, T, C.
Why are we using transgenic mouse
models?
Cell types
Goal 1: Overexpression of proteins
Goal 2: Try to catch someones eye – Use of fluorescent proteins
J
Goal: Try to catch so.’s eye in CNS J
Chemistry Nobel Prize 2008
• Osamu Shimomuto
• Martin Chalfie
• Roger Y. Tsien
GFP – Aquorea victoria
1. Publication in 1997 with cell type specific expression in mice
Nowadays: uncountable!!!
Use of fluorescent proteins !!!
ECFP - astrocytes
EGFP - astrocytes
DsRed1 oligodendrocytes
EYFP - neurons
AmCyan - astrocytes
mRFP1
astrocytes
Neuron glia interaction in transgenic modified animals I
interaction in transgenic modified animals III
Transgenic mice
TgN()
TgN: transgene non homologous recombination
TgH()
TgH: transgene homologous recombination
TgR()
TgR: retroviral transgene insertion
Use of wild type mice and viruses
(rAAVs, Lentiviruses…)
Why are we using inbred mouse strains for
research?
Excurs I: Wild type mouse strains
Inzucht oder Auszucht?
• Inzucht: C57Bl/6N, C57Bl/6J, FVB/N, Balb/c, DBA,
C3H, …
• Auszucht: NMRI, CD-1, ICR, …
à Unterschiedlicher Preis
Excurs I: Inbred strains
Definition Inzucht: (Jackson lab, Harlan etc.):
• mind. 20 Generationen konsequenter VP von Wurfgeschwistern (Brüder &
Schwestern)!!!
• starke Selektion der Zuchttiere (70% nicht verwendet, Purging)
• 20ste Generation: 98,4 - 99,4% homozygot (reinerbig)
• 1998: Jackson lab: 436 Mausinzuchtstämme
• Oft mit mehr als 150 Generationen reiner Inzucht
WARUM???
Vorteil: à hohe genetische Einheitlichkeit/stammspezifische Besonderheiten
à weniger Tiere, trotzdem reproduzierbare Ergebnisse!!!
Excurs I: Inbred strains
Problem: Inzucht-Depression
Def:
reduzierte biologische Fitness in gegebener Population als Resultat
der Inzucht
à geringere Fertilität, Tiere kleiner, höhere Anfälligkeit gegenüber
Krankheiten etc.
à unerwünschter genetischer Drift
à Purging-Selektion: Phänotypen der schädlichen rezessiven Allele bei
Inzucht exponiert und ausselektiert (PCR Marker)
Excurs I: Outbred strains
Auszucht:
à genutzt, wenn genetische Diversität in Testpopulation erwünscht ist
oder Preis für Tiere gering sein muss
à häufig in biologischen Assays/Vorstudien zur Etablierung von
Techniken verwendet (bevor dann eigentl. Experimente mit
Inzuchtstämmen durchgeführt werden)
à Nutzung, wenn hohes Körpergewicht oder gutes Zuchtverhalten
wichtiger sind als definierter GT
à oft als Ammen verwendet (NMRI)
à robust, hohe Reproduktion (grosse Wurfzahlen)
Haltung:
• als geschlossene Kolonie genetische variabler Zusammensetzung
• mind. 200 Weibchen als Zuchtstamm (Harlan)
Excurs I: Outbred strains
à Vermehrung von miteinander nicht-verwandten Zuchtpaaren
à Erhaltung der stammspezifischen Mischerbigkeit (Heterozygotie)
à Angaben zu min. Zuchtpaaren immer unterste Grenze
à geschlossene Kolonien, ohne Zufuhr von Aussen
à Häufig bei Tieren mit längerer Generationsfolge (Hund, Katze,
Kaninchen)
Excurs I: Outbred strains
1) Zufallspaarung (Panmixie)
à zufällige VP, mind. 100 Zuchtpaare
à verschiedene methodische Mängel, nur in Ausnahmefällen
angewendet
2) Rotationsverpaarung
à Mindestansatz 25 VP, Unterteilung
der Populationen in Blöcke
à pro Generation starres VP-Schema
zwischen Blöcken
à WICHTIG: ständiges Einhalten von
Blockzahl und VP-Rhythmus
3) Pedigreezucht
à min. 10 ZP, geeignet für grössere Versuchstierarten
(Hund, Katze, Schwein)/seltene Versuchstiere
à Verpaarung anhand der Abstammung nach möglichst
geringem Verwandtschaftsgrad
à Zuchtpaar mit max. einem gemeinsamen Urgrosselter
TgN(hGFAP-mRFP1)GRFT
TgN: transgene non homologous recombination
TgH(NG2-CreERT2)NGCE
TgH: transgene homologous recombination
Definition gene:
Gen:
à Abschnitt auf der DNA mit Grundinformation zur Herstellung
einer biologisch aktiven Ribonukleinsäure (RNA)
à 3‘ oder 5‘-Richtung – regulatorische Sequenzen
intron
DNA
Promoter
exon1
intron
intron
exon 3
ex2
Transkription (Abschreiben)
prämRNA
intron
exon1
intron
ex2
intron
exon 3
Reifung (Herausschneiden der Introns)
reife mRNA
Translation (Übersetzen)
Protein
exon1 ex2
exon 3
AAAAAA
Cloning strategy I - TgN
•
•
•
•
Cloning of transgene
Injection of linearised DNA construct
Genotyping (PCR of DNA of born pups)
Founder analysis of PCR positive animals
à identification of transgene expression founder
• Breeding
• Characterisation of founder(s)
à Research
Non homologous recombination – simplest strategy
Non homologous recombination – cloning
Non homologous recombination –
Microinjection of linearised DNA in oocytes
Non homologous recombination –
Microinjection of linearised DNA in oocytes
Random insertion of transgene
=>
Copyright 2004 University of Washington, Department of Pathology
10
Integration takes place
Control 2
control 1
sense
wild type
H2O
Animal 5
Animal 4
animal 3
animal 2
animal 1
Non homologous recombination – PCR analysis
antisense
PCR TgN(hGFAP-ECFP) - non homologous recombination
Solution – control band
Pyrat genotypes
wt
primer1 - located in Promoter (blue)
primer 2 - located in XFP (blue)
xfp/wt
no band - wt or no DNA
one band - het or hom, no difference in PCR!!!
GFAP ECFP
GFAP ECFP
xfp
PCR example
xfp
wt
no DNA
xfp
wt
à addition of DNA loading control
xfp/xfp
(independent wt gene) (red )
no band - no DNA
GFAP ECFP
only loading control band- wt
two bands (loading control and transgene - transgenic animal (hom
or het)
xfp
xfp
PCR example
wt
no DNA
xfp
wt
PCR itself: no visible difference!!! à quantitative Realtime-PCR
hom vs. het???
à Quantitative Realtime-PCR
Non homologous recombination – Founder analysis
Founder I
Founder II
Founder III
Non homologous recombination – Founder analysis
Short reminder:
You start with ONE founder, technically always inbreeding!!!
perfect: founder is male, female less efficient!!!
Brazilian mice – used in our group
green
blue
Bergmann Glia
microglia
yellow
neurons
Summary I
•
•
•
•
Cloning of transgene
Injection of linearised DNA construct
Genotyping (PCR of DNA of born pups)
Founder analysis of PCR positive animals
à identification of transgene expression founder
• Breeding
• Characterisation of founder(s)
à Research
Cloning strategy II
•
•
•
•
•
•
Cloning of transgene
Homologous recombination in ES cells
Identification of ES cell clone
Injection of ES cells in blastocysts
Identification of chimeras
Breeding
à Research
Embryonic stem cells –
stable lines from diverse inbred strains available
Einzeller (Zygote)
Tag = 0.5
Zweizeller
Tag = 1.5
8-Zeller/Morula
Tag = 2.5
Innere Zellmasse
Blastozyste
Tag = 3.5
Embryonale Stammzellen
Electroporation
• mixture of transgenic DNA construct and ES cells
à electroshock
• unspecific uptake of DNA in ES cells
Homologous recombination in ES cells
DNA in Nucleus
X
X
Homologous recombination
Integration takes place
gene for resistance against antibiotics
Homologous recombined cells
Cells with randomly integrated DNA
à all with resistance against antibiotics
Antibiotics selection
Only cells with integrated DNA survive and proliferate
à Pick the clones and check them (sequencing)!!!
antibiotics
?
Blastocyst injection
Chimera
ES cell injection - blastocytes
Germ line integration
x
à Successful establishment of new transgenic mouse line
generated with homologous recombination
à one chimera as start à inbreeding can‘t/won‘t be avoided
Example 1: 129Sv in C57Bl/6N
blastocysts:
C57Bl/6N (Tyr+/ Tyr+, a/a)
ES cells:
129Sv (Tyr+/ Tyr+, A/A)
x
Chimera
Tyr+/ Tyr+, A/a
Tyr+/ Tyr+, a/a
A = agouti dom.
A = agouti rez.
Homologous recombination
wt
exon 1
exon 2
KI
EGFP
exon 1
exon 2
KO
exon 1
exon 2
cKO
exon 1
exon 2
Homologous recombination
wt
exon 1
exon 2
KI
EGFP
exon 1
exon 2
Microglial cells are highly dynamic at rest (WM)
TgH(CX3CR1-EGFP)
Steffen Jung, Dan Littman
185 min, 5 min interval
Immediate responses of microglia to microlesion contrary to astrocytes
microglia (EGFP)
astrocytes (TexasRed)
TgH(CX3CR1-EGFP)
Homologous recombination
wt
exon 1
exon 2
KO
exon 1
exon 2
Problem: KO in all cells of the organism
à Cav, Nav etc.
Solution: conditional KO (use of Cre DNA recombinase and it‘s
inducible varients CreER, CreERT, CreERT2 etc.
Homologous recombination
wt
exon 1
exon 2
cKO
exon 1
exon 2
+ Cre
Important:
1) two mouse lines: a) floxed line
b) cre driver line
2) loxP sites in introns (w/o cre like wt mice)
closer view to conditional KO’s – cell type specific
cKO
exon 1
promoter
cells
without Cre
exon 1
exon 2
exon 2
Cre
cells
with Cre
Cre vs. CreERT2 (cell type specificity + control of time)
cKO
exon 1
exon 2
promoter
Cre
cells
without Cre
exon 1
exon 2
exon 1
cKO
promoter
cells
with Cre
exon 2
CreERT2
cells
without CreERT2
exon 1
cells
with
CreERT2
exon 2
exon 1
cells
without CreERT2
exon 1
exon 2
exon 2
cells
with
CreERT2
Homologous recombination – construct
Huang et al., Glia, 2014
Homologous recombination – reporter expression
Huang et al., Glia, 2014
PCR TgH(CX3CR1-EGFP) - homologous recombination - KI
Pyrat genotypes
wt
xfp/wt
For example:
primer 1 - sense - located in wildtype(blue)
primer 2 - antisense - located in wildtype (red)
primer 3 - antisense - located in knockin (green)
EGFP
xfp/xfp
PCR example
EGFP
xfp/wt
xfp/wt
wt
no DNA
xfp/xfp
xfp/xfp
EGFP
wt
wt
xfp/xfp
xfp/wt
xfp/xfp
one band with a size of wt - wt animal
one band with a size of KI - homozygous knockin
Two bands – heterozygous animal
à NO DNA loading control
Example MolPhys
P2Y1
exon 1
exon 2
GCaMP3
Stop cassette
GCaMP3
GLASTCreERT2
Glast locus
CreERT2
à Tamoxifen inducible P2Y1 KO in astrocytes, KO cells express GCaMP3!!!
(simple example, more complicated possible à database necessary (PyRAT)
In vivo 2P-LSM in awake mice (ctx and cb)
50 µm
Ca2+ imaging
Unpublished Molecular Physiology
Summary II
•
•
•
•
•
•
Cloning of transgene
Homologous recombination in ES cells
Identification of ES cell clone
Injection of ES cells in blastocysts
Identification of chimeras
Breeding
à Research
All transgenic animals follow mendelian inheritance
xfp/wt cross xfp/ wt
à25% xfp/xfp
à50% xfp/wt
à25% wt
BUT: don‘t forget littersize in breeding plans
Examples for transgenic expression/recombination
• Comparison between TgH and TgN – What do
you choose?
• Does the background strain of transgenic mice
influence transgene expression?
TgH vs TgN
TgH(Glast-CreERT2)
TgN(hGFAP-CreERT2)
• real KI à expressed in all
cells with slc1a3 activity
• Overexpression
• but: not expressed in all
cells with mGFAP promoter
activity
Mori et al., 2006 Glia
Hirrlinger et al., 2006 Glia
TgH vs TgN
TgH(hGlast-CreERT2)
• used in many different publications (mainly lineage studies)
• all astrocytes recombine (total astrocyte KO possible)
• no difference between recombination in males and females
kindly provided by Laura Schlosser, unpublished
TgH vs TgN
TgN(hGFAP-CreERT2)
• used in many different publications (as in Lioy et al., Nature
2011, Han et al., 2012, Cell etc)
• not all astrocytes recombine (KO vs. rescue)
• difference between recombination in males and females
kindly provided by Xianshu Bai, unpublished
TgH vs TgN
TgH(Glast-CreERT2)
• used for many studies
• example: conditional KO of
AMPA-R in BG cells
• mice lack AMPA-R in BG
and additionally on Glast
allele
Saab et al., Science 2012
conclusion:
à KI gives you all cells of interest, but half a KO
à TgN: not all cells, overexpression can lead to
activation of endogenous proteins
à always controls necessary
Examples for transgenic expression/recombination
• Comparison between TgH and TgN – What do
you choose?
• Does the background strain of transgenic mice
influence transgene expression?
mice
FVB/N (FVB)
C57Bl/6N
(B6N)
Is the background of a mouse strain important for
the expression of transgenic proteins?
à Both inbred strains
à Bai et al., 2013 (PLoS One)
Constructs/mouse lines (TgN)
Focus: cerebellum
>N12
Histological and biochemical analysis
Bai et al., 2013
conclusion:
à genetic background affects human glial fibrillary
acidic protein promoter activity
à always controls necessary
à not known for other frequently used promoters
Conclusions
Conclusion:
• mostly used lab mice are inbred strains
• different time durations for generation of TgN and
TgH mice
• important differences in TgN and TgH for
experiments (alleles, overexpression etc.) as well as
maintenance (genotyping etc.)
• transgenic mice follow Mendelian rules
• PyRAT database
Questions?
Anja Scheller
Molecular Physiology
Institute of Physiology
Medical School
University of Saarland
[email protected]
[email protected]