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Homologous recombination
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]
