Molecular Biology and Genetics

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 10 No. 2, Issue of January 15, 2007
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received December 14, 2005 / Accepted October 26, 2006
DOI: 10.2225/vol10-issue2-fulltext-1
SHORT COMMUNICATION

Cre-loxP recombination vectors for promoter studies

Nina Pedersen
Department of Radiation Biology
Section 6321
National University Hospital
Blegdamsvej 9, 2100 Copenhagen Ø
Denmark
Tel: 45 35 45 63 09
Fax: 45 35 45 63 01
E-mail: nina.pedersen@rh.hosp.dk

Thomas Tuxen Poulsen
Department of Radiation Biology
Section 6321
National University Hospital
Blegdamsvej 9, 2100 Copenhagen Ø
Denmark
Tel: 45 35 45 63 28
Fax: 45 35 45 63 01
E-mail: tuxen@rh.dk

Hans Skovgaard Poulsen*
Department of Radiation Biology
Section 6321
National University Hospital
Blegdamsvej 9, 2100 Copenhagen Ø
Denmark
Tel: 45 35 45 63 03
Fax: 45 35 45 63 01
E-mail: hans.skovgaard.poulsen@rh.hosp.dk

Website: www.radiationbiology.dk

*Corresponding author

Financial support: National University Hospital, the Danish Cancer Society, the Danish Medical Research Council, TheDanish Cancer Research Foundation, the Erik Hørslev og Hustru Birgit Hørslevs Foundation, the Aase and Ejnar Danielsens Foundation and the A.P. Møller Foundation for the Advancement of Medical Science.

Keywords: cloning, Cre recombinase, plasmid, reporter genes, therapeutic genes.

Abbreviations:

cDNA: complementary DNA
EGFP: enhanced green fluorescent protein
HSV-TK: Herpes Simplex virus thymidine kinase
PCR: polymerase chain reaction
SNP: single nucleotide polymorphism

Abstract
Full Text

For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning.

In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding.

We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.

Supported by UNESCO / MIRCEN network