IOSI Journal Club Giulia Poretti June 1, 2007

1. IOSI Journal Club Giulia Poretti June 1, 2007

2. RMCE targeted transgenesis system in a lymphoma cell line:a tool for studying the function of candidate genes

3. RMCERecombinase-mediated cassette exchange A site-specific system for single copy integration of a transgene • Two-step procedure: • Recognition sites for a site-specific recombinase (e.g. Cre) are targeted to the locus of interest by homologous recombination or inserted at random by illegitimate recombination. → Creation of a cassette with reporter gene (selection cassette) flanked by two recognition sites at the integration site Modified from Baer A et al. Curr Opin Biotechnol. 2001;12:473-480

4. RMCERecombinase-mediated cassette exchange A site-specific system for single copy integration of a transgene • Two-step procedure: • Recognition sites for a site-specific recombinase (e.g. Cre) are targeted to the locus of interest by homologous recombination or inserted at random by illegitimate recombination. → Creation of a cassette with reporter gene (selection cassette) flanked by two recognition sites at the integration site 2) A new sequence with analog flanking recognition sites presented on a targeting vector replaces the resident cassette by site-specific recombinase-mediated cassette exchange.

5. RMCERecombinase-mediated cassette exchange Modified from Baer A et al. Curr Opin Biotechnol. 2001;12:473-480

6. in vivo model:Granta-519cellular model system for Mantle Cell Lymphoma (MCL)

7. CDKN2A/B deletion Granta-519: structural rearrangements • Genomic profile:

8. CDKN2A/B deletion Granta-519: structural rearrangements • Genomic profile: • Translocation: t(11;14) CCND1 overexpression

9. RP11-149I2 Genomic complementation of inactivated tumor suppressor genes Inactivated tumor suppressor gene: CDKN2A/B (cyclin-dependent kinase inhibitor 2A/B) Genomic DNA insert: RP11-149I2

10. Bacterial Artificial Chromosome (BAC) • Synthetic DNA molecule representing large segments of human genomic DNA (complexity reduction) • Contains also bacterial DNA sequences needed for replication and segregation in bacteria • One important application of BAC libraries is their use for sequencing the complete human genome • see YAC (Yeast Artificial Chromosome): yeast chromosome into which foreign DNA (up to 1000 kb) has been inserted for replication in dividing yeast cells

11. shRNA-CCND1 Gene silencing of activated oncogenes Activated oncogene: CCND1 cyclin D1 CCND1-specific shRNA coding insert: shRNA-CCND1 Controls: empty vector unspecific-shRNA

12. Targeting vector with selection cassette flanked by recognition sites for site-specific recombination (recombinase Cre) is integrated randomly into the genome of Granta-519 Site-specific integration of transgene (I) • Transfected cells are positively selected by neomycin resistance

13. Integration sites ofselection cassetteFISH • Three monoclonal Granta-519 sublines were isolated with different integration sites for the selection cassette: 18q (G18), 11q (G11), 10p:

14. 10p 18q Integration sites of selection cassette:FISH • Three monoclonal Granta-519 sublines were isolated with different integration sites for the selection cassette: 18q (G18), 11q (G11), 10p: (B) Granta 519 subclone G18

15. 11q Integration sites of selection cassette:FISH Granta 519 subclone G11: integration site on 11q

16. Transfected cells are positively selected by neomycin resistance • Co-transfection of Cre-expression plasmid (pCMXhCre) + OR knock down CCND1 knock-in CDKN2A/B Site-specific integration of transgene (I) • Targeting vector with selection cassette flanked by recognition sites for site-specific recombination (recombinase Cre) is integrated randomly into the genome of Granta-519

17. shRNA-CCND1 • Transfected cells are positively selected by neomycin resistance • Co-transfection of Cre-expression plasmid (pCMXhCre) + OR RP11-149I2 knock down CCND1 knock-in CDKN2A/B Site-specific integration of transgene (I) • Targeting vector with selection cassette flanked by recognition sites for site-specific recombination (recombinase Cre) is integrated randomly into the genome of Granta-519

18. Site-specific integration of transgene (II) • The selection cassette between the loxP-sites is substituted by RMCE with a single copy of the cloned insert OR • The new subclones are negatively selected by ganciclovir

19. shRNA-CCND1 RP11-149I2 Site-specific integration of transgene (II) • The selection cassette between the loxP-sites is substituted by RMCE with a single copy of the cloned insert OR • The new subclones are negatively selected by ganciclovir

20. Site-specific integration:RP11-149I2FISH 11q BAC clone selection cassette replaced by transgene RP11-149I2 on 11q

21. Site-specific integration: sh-RNA-CCND1genomic PCR

22. RMCE targeted transgenesis vsDNA transfection DNA trasfection • Transfected DNA forms a large repeating unit of tandem repeats • Transfected unit is unstable unless it becomes integrated into a host chromosome by nonhomologous recombination transient transfectans:transfected DNA remain in extrachromosomal form stable transfectans: transfected DNA is integrated into the genome • Expression of transfected DNA possible both in transient and in stable transfectants but unpredictable (random insertion) • The arrangement of transfected DNA sequences is different in each transfected line, but remains constant during propagation of that line

23. RMCE targeted transgenesis vsDNA transfection RMCE targeted transgenesis • A single copy of the transgene is integrated in the genome of the recipient • Targeted integration of the transgene in precharacterized chromosomal site • Transfected unit is stable • The insertion of transfected DNA sequences is targeted and constant in each transfected line • BAC DNA inserts: expression of the transgene according to „wild-type“ regulatory elements (e.g. promoters, enhancers) • Overcome the limited transfection efficiency of B cell lymphocytes, particularly difficult to transfect if usign BAC DNA

24. Validation of specific gene activity modulation

25. CDKN2A/BKnock-in at RNA level: RT-PCR

26. CDKN2BKnock-in at protein level: IHC

27. CCND1Knock down at RNA level: RT-PCR

28. CCND1Knock down at protein level: western blot

29. Proliferation assay by MTS

30. Discussion

31. Key experiment

32. Strongest point

33. Take home message • Targeted integration of transgene via RMCE allowed the modulation of gene activity counteracting the deregulation given by oncogenic processes such as inactivation of tumor suppressor gene and activaton of oncogene. • Advantages of targeted integration into the recipient genome vs DNA transfection

35. Paper discussion • What is the key experiment? (the one confirming the statement in the title) • What is the strongest point? • What is the weakest point? and What to do to strenghten it? • What is the take home message? (summarize it in a sentence)

36. RMCERecombinase-mediated cassette exchange Baer A et al. Curr Opin Biotechnol. 2001;12:473-480

37. RMCERecombinase-mediated cassette exchange • well suited for the rapid generation of multiple mutant alleles of a given genomic locus • low efficiency in the absence of selection and due to the promiscuity of the participating recombinase recognition sites: use two independent recombinase systems (e.g. loxP on one and FRT on the other side of the cassette together with a Cre/Flpe expression vector) Matthias Lauth et al. NAR, 2002

38. RMCERecombinase-mediated cassette exchange • recombinase-mediated cassette exchange (RMCE) techniques which cleanly replace a resident cassette that is flanked by two hetero-specific recombination target sites for a second cassette with the analogous design, presented on a targeting vector. Bode,J et al. (2000) Biol. Chem., 381, 801–813.[ISI][Medline]