iosi journal club giulia poretti june 1, 2007
DESCRIPTION
IOSI Journal Club Giulia Poretti June 1, 2007. RMCE targeted transgenesis system in a lymphoma cell line: a tool for studying the function of candidate genes. RMCE Recombinase-mediated cassette exchange. A site-specific system for single copy integration of a transgene Two-step procedure: - PowerPoint PPT PresentationTRANSCRIPT
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IOSI Journal Club
Giulia PorettiJune 1, 2007
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RMCE targeted transgenesis system in a lymphoma cell line:
a tool for studying the function of candidate genes
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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
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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.
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RMCE Recombinase-mediated cassette exchange
Modified from Baer A et al. Curr Opin Biotechnol. 2001;12:473-480
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in vivo model:
Granta-519
cellular model system for Mantle Cell Lymphoma (MCL)
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Granta-519: structural rearrangements
CDKN2A/B deletion
• Genomic profile:
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Granta-519: structural rearrangements
• Translocation: t(11;14) CCND1 overexpression
CDKN2A/B deletion
• Genomic profile:
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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
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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
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Gene silencing of activated oncogenes
Activated oncogene: CCND1 cyclin D1
CCND1-specific shRNA coding insert: shRNA-CCND1
Controls: empty vector unspecific-shRNA
shRNA-CCND1
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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
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Integration sites of selection cassetteFISH
• Three monoclonal Granta-519 sublines were isolated with different integration sites for the selection cassette:18q (G18), 11q (G11), 10p:
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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
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Granta 519 subclone G11: integration site on 11q
Integration sites of selection cassette:FISH
11q
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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 CCND1knock-in CDKN2A/B
OR
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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 CCND1knock-in CDKN2A/B
OR
shRNA-CCND1RP11-149I2
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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
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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
RP11-149I2 shRNA-CCND1
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Site-specific integration: RP11-149I2 FISH
selection cassette replaced by transgene RP11-149I2 on 11q
11q BAC clone
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Site-specific integration: sh-RNA-CCND1genomic PCR
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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 recombinationtransient 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
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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
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Validation
of
specific gene activity modulation
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CDKN2A/BKnock-in at RNA level: RT-PCR
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CDKN2BKnock-in at protein level: IHC
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CCND1Knock down at RNA level: RT-PCR
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CCND1Knock down at protein level: western blot
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Proliferation assay by MTS
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Discussion
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Key experiment
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Strongest point
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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
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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)
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RMCE Recombinase-mediated cassette exchange
Baer A et al. Curr Opin Biotechnol. 2001;12:473-480
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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
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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]