Cyr61 knockout mice

Cyr61 is a member of the recently identified CCN family of angiogenic regulators. This family of secreted proteins consists of six members: Cyr61, CTGF, Nov, WISP-1, WISP-2, and WISP-3. These proteins share 4 modular domains with sequence similarities to insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and growth factor cysteine-knots. Cyr61 is encoded by immediate-early gene and is co-induced by serum, bFGF, PDGF, and TGF-ß1 in fibroblasts. It associates with the extracellular matrix. Purified Cyr61 mediates cell adhesion, stimulates cell migration, and augments growth factor-induced DNA synthesis. In vitro, Cyr61 can promote chondrogenic differentiation, consistent with their expression in prechondrogenic mesenchyme during embryogenesis. It stimulates chemotaxis in endothelial cells through an integrin a v ß 3 -dependent pathway, and induces neovascularization in vivo. Expression of Cyr61 in tumor cells enhances tumorigenicity by increasing tumor size and vascularization. Cyr61 has also been shown to be induced in granulation tissues during cutaneous wound healing. Targeted gene disruption experiments in whole organisms have not been reported for members of the CCN gene family to date. It is therefore the aim of this proposal to study the effect of the knockout of Cyr61 in a mouse model. It is rather likely that a "classical" Cyr61 gene knockout might result in embryonic lethality, thus impairing studies of some of the proposed functions. Therefore we aim to study Cyr61 function using a conditional gene knockout approach that will enable to deactivate its expression specifically in selected tissues at certain stages of ontogeny. So far, a gene encoding Cyr61 was isolated and extensively characterized and targeting vector for conditional Cyr61 gene inactivation was prepared. Additionally, in in vitro conditions, using a purified Cre recombinase, we have shown that the most critical component of this system - Cre-loxP system is functioning. This vector will be used to electroporate R1 embryonic stem (ES) cells and correct homologously recombined clones will be used to generate Cyr61 deficient mice. Obtained heterozygous and homozygous transgenes will be further crossed either with transgenic mice tissue- specifically expressing Cre protein under the control of a Tie-1 promoter or with mice in which Cre protein is expressed ubiquitously, but is activated only with 4-hydroxytamoxifen, thus allowing to disrupt Cyr61 in tissues where Cre protein is activated. Successful solution of the proposed project should further elucidate the anticipated role of this protein in development, chondrogenesis, vasculogenesis and also its possible involvement in wound healing and tumorigenesis.

The use of transgenic animals has dramatically increased during the last decade and it has greatly advanced our knowledge of gene regulation and function, mammalian development and pathogenesis of human disease. Using an embryonic stem (ES) cells gene technology, it became possible to deactivate specific genes in mice, thus obtaining relevant information about the function of the gene of interest. Some of the gene inactivations result in embryonic lethalities, thereby hampering to obtain relevant information about the function of gene of interest in adult animals. This fully concerns to inactivation of Cyr61 gene where Cyr61-null mice suffered from embryonic death due to compromised blood vessel integrity and placental vascular insufficiency. In order to reveal function of Cyr61 in postnatal processes, within this project we generated mice "with conditional Cyr61 inactivation" that enabled us to inactivate Cyr61 in mice during the postnatal period only. To this end, we cloned Cyr61 gene and generated so called targeting vector. Introduction of this vector into the ES cells by electroporation resulted in the replacement of the endogenous Cyr61 gene by almost identical Cyr61 gene, but differing by the presence of "loxP sites". In those cells when Cre protein is present, loxP sites will be recognized and the sequence flanked by these sites will be deleted. We aggregated such modified ES cells with embryos isolated from wild-type mothers, transferred them into hormonally prepared mothers. Thus we obtained several highly chimeric mice that were able to transfer such modified genetic information to their offspring. Such mice were subsequently crossed with transgenic mice expressing Cre protein upon 4-hydroxytamoxifen administration. Development of established double-transgenic mice was normal, but upon 4-hydroxytamoxifen administration led to Cyr61 inactivation during the postnatal period and enabled us to study consequences of such inactivation. We could show that Cyr61 plays important role in wound healing and its inactivation leads to the delay of this process. Further analyses will reveal the Cyr61 function in generation of new vessels in postnatal mice, in restenosis and in female reproduction. These studies are currently in progress and will be accomplished within this project. Since many of physiological processes in mice and human are analogous, upon project completion we can draw conclusions about physiological role of Cyr61 in man.