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. RMCE Recombinase-mediated cassette exchange A site-specific system for single copy integration of a transgene Two-step procedure: 1)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. RMCE Recombinase-mediated cassette exchange A site-specific system for single copy integration of a transgene Two-step procedure: 1)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. RMCE Recombinase-mediated cassette exchange Modified from Baer A et al. Curr Opin Biotechnol. 2001;12:473-480

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

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

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

9. Genomic complementation of inactivated tumor suppressor genes Inactivated tumor suppressor gene: CDKN2A/B (cyclin-dependent kinase inhibitor 2A/B) Genomic DNA insert: RP11-149I2 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. Gene silencing of activated oncogenes Activated oncogene: CCND1 cyclin D1 CCND1-specific shRNA coding insert: shRNA-CCND1 Controls:empty vector unspecific-shRNA shRNA-CCND1

12. 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 Transfected cells are positively selected by neomycin resistance

13. 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:

14. 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: 10p 18q (B) Granta 519 subclone G18

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

16. 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 Transfected cells are positively selected by neomycin resistance Co-transfection of Cre-expression plasmid (pCMXhCre) + knock down CCND1 knock-in CDKN2A/B OR

17. 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 Transfected cells are positively selected by neomycin resistance Co-transfection of Cre-expression plasmid (pCMXhCre) + knock down CCND1 knock-in CDKN2A/B OR shRNA-CCND1RP11-149I2

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 The new subclones are negatively selected by ganciclovir OR

19. 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 The new subclones are negatively selected by ganciclovir OR RP11-149I2shRNA-CCND1

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

21. Site-specific integration: sh-RNA-CCND1 genomic PCR

22. RMCE targeted transgenesis vs DNA 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 vs DNA 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/B Knock-in at RNA level: RT-PCR

26. CDKN2B Knock-in at protein level: IHC

27. CCND1 Knock down at RNA level: RT-PCR

28. CCND1 Knock 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. RMCE Recombinase-mediated cassette exchange Baer A et al. Curr Opin Biotechnol. 2001;12:473-480

37. RMCE Recombinase-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. RMCE Recombinase-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][ISI][Medline]