Generation of mice with mutagenized u-pa genes, via the cre/lox recombination strategy

Z. Liang, E. Rosen, P. Carmeliet, F. Castellino, D. Collen

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Abstract

Urokinase type plasminogen activator (u-PA) converts the zymogen plasminogen into the protease plasmin. Plasmin activity is involved in several important biological processes including fibrinolysis, cell migration, tissue remodelling and metastasis. u-PA is a 54kd protein containing distinct growth factor (GDF), kringle and protease domains. The GFD, encoded by exon 4, mediates the binding of u-PA to its cellular receptor u-PAR and this interaction is thought to be important for u-PA mediated biological processes. To study the biological role of the u-PA/u-PAR interaction, targeted mutations of the u-PA gene deleting the GFD are constructed using the Crelox system. This involves the use of the Cre recombinase which mediates site specific recombination between two 34 bp lox sites, removing the sequences between the two sites. A modified u-PA gene in which exon 4 was replaced with a iox-neo-tklox cassette was introduced into embryonic stem cells (ES cells) and substituted for a wild-type copy of the u-PA gene by homologous recombination. After eleclroporation with a plasmid expressing the Cre recombinase, site specific recombination between the two lox sites looped out the neo and Ik genes. Sequence analysis of the modified gene confirmed that the u-PA gene rearranged as expected generating a u-PA GFD allele. To determine if the modified gene was transcribed and spliced as expected, RNA was isolated from cystic embroid bodies (CEBs) developed from the u-PA GFD ES cells. Analysis by rtPCR using exon 1 and exon 11 primers produced the expected two u-PA bands of 1.4 and 1.2 kb. Both bands hybridized to exon 3 and exon 5 probes, while only the larger band hybridized to an exon 4 probe. These results indicate that the modified gene was spliced as expected. The u-PA GFD ES cells have been introduced into mouse embryos by embryo aggregation to generate u-PA GFD mice. A similar strategy is being used to generate mice with a proteolytically inactive urokinase gene.

Original languageEnglish (US)
Number of pages1
JournalFibrinolysis
Volume10
Issue numberSUPPL. 3
StatePublished - Dec 1 1996

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ASJC Scopus subject areas

  • Hematology

Cite this

Liang, Z., Rosen, E., Carmeliet, P., Castellino, F., & Collen, D. (1996). Generation of mice with mutagenized u-pa genes, via the cre/lox recombination strategy. Fibrinolysis, 10(SUPPL. 3).