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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
•
1)
→
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
RMCE
Recombinase-mediated cassette exchange
•
1)
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.
RMCE
Recombinase-mediated cassette exchange
Modified from Baer A et al. Curr Opin Biotechnol. 2001;12:473-480
in vivo model:
Granta-519
cellular model system
for Mantle Cell Lymphoma (MCL)
Granta-519: structural rearrangements
• Genomic profile:
CDKN2A/B deletion
Granta-519: structural rearrangements
• Genomic profile:
CDKN2A/B deletion
• Translocation: t(11;14) CCND1 overexpression
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
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
Gene silencing of
activated oncogenes
Activated oncogene: CCND1 cyclin D1
CCND1-specific shRNA coding insert: shRNA-CCND1
shRNA-CCND1
Controls:
empty vector
unspecific-shRNA
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
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:
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
Integration sites of selection cassette:
FISH
11q
Granta 519 subclone G11: integration site on 11q
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) +
OR
knock-in CDKN2A/B
knock down CCND1
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) +
OR
knock-in CDKN2A/B
RP11-149I2
knock down CCND1
shRNA-CCND1
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
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
RP11-149I2
shRNA-CCND1
• The new subclones are negatively selected by
ganciclovir
Site-specific integration: RP11-149I2
FISH
11q
BAC clone
selection cassette replaced by transgene RP11-149I2 on 11q
Site-specific integration: sh-RNA-CCND1
genomic PCR
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
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
Validation
of
specific gene activity modulation
CDKN2A/B
Knock-in at RNA level: RT-PCR
CDKN2B
Knock-in at protein level: IHC
CCND1
Knock down at RNA level: RT-PCR
CCND1
Knock down at protein level: western blot
Proliferation assay by MTS
Discussion
Key experiment
Strongest point
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
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)
RMCE
Recombinase-mediated cassette exchange
Baer A et al. Curr Opin Biotechnol. 2001;12:473-480
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
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]