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:

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 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/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]