IOSI Journal Club

Giulia PorettiJune 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: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

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.

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

CDKN2A/B deletion

• Genomic profile:

Granta-519: structural rearrangements

• Translocation: t(11;14) CCND1 overexpression

CDKN2A/B deletion

• Genomic profile:

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

Controls: empty vector unspecific-shRNA

shRNA-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

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:

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

Granta 519 subclone G11: integration site on 11q

Integration sites of selection cassette:FISH

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) +

knock down CCND1knock-in CDKN2A/B

OR

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

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

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

Site-specific integration: RP11-149I2 FISH

selection cassette replaced by transgene RP11-149I2 on 11q

11q BAC clone

Site-specific integration: sh-RNA-CCND1genomic 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 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

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/BKnock-in at RNA level: RT-PCR

CDKN2BKnock-in at protein level: IHC

CCND1Knock down at RNA level: RT-PCR

CCND1Knock 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]