DNA vector for bone-specific gene expression

Details DNA vector for bone-specific gene expression DNA vector for bone-specific gene expression

1997-06-17

1999-09-07

Campell, Bruce R.

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

800DIG1, 424 92, 435 29, 4353201, 435325, 435354, 935 6, 935 34, 935 59, C12N 500, C12N 1500, A61K 4900

Patent

active

059489510

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to an expression vector for use in producing transgenic animals, in particular transgenic mice. The present invention further relates to transgenic animals including this vector.
Osteoporotic fractures are a large and growing health care problem. The recent finding that alleles of the vitamin D receptor (VDR) gene predict bone mineral density is the first direct molecular demonstration of a genetic contribution to the risk for osteoporosis. Recent investigations have demonstrated the VDR allelic effect on intestinal calcium absorption and fracture risk. These differences in VDR gene alleles thus predict differences in bone turnover and bone mass associated with differences in bone and calcium homeostasis. Studies of human subjects, though informative, are not adequate to define the mechanism by which the VDR allelic effect is realised. The proposed studies utilise transgenic mice to address the hypothesis that modest alterations in VDR protein level will affect bone turnover.
Osteocalcin is the most abundant non-collagenous protein in bone. Molecular studies of the human osteocalcin gene have revealed a number of response elements to different nuclear transcription factors, including the vitamin D receptor and its ligand 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To date these studies have concentrated on regions 5' of the osteocalcin locus, particularly those regions within 1300 bp of the transcription start site that contains the VDRE. Efforts however to utilise these regions to direct tissue specific gene expression in bone have not been successful with gene expression in ectopic tissue (Kesterson et al Molec Endocrin 7 462-467, 1993). Transgene expression has also been at low levels in bone indicating that other cis acting elements are required for full expression of transgenes in a tissue specific and copy number dependent manner. Regions 3' and further 5' of the human osteocalcin gene were therefore examined for their role in regulating bone specific gene transcription, initially via transient transfection of CAT reporter constructs into the osteoblast-like cell line ROS 17/2.8, and then after stable integration of the transgene into the inbred FVB/N mouse genome.
3.8 kb of the 5' flanking sequence cloned into a CAT expression vector (OSCAT 6) increases basal activity (2.6+0.8 fold) when compared to a smaller 1.3 kb 5' fragment (OSCAT 2) known to contain the vitamin D response element (VDRE). However, fold induction seen in OSCAT 2 by 10.sup.-8 M 1,25-(OH)2D3, is identical to that observed for the OSCAT 6 construct.
These data indicate the existence of previously uncharacterised regulatory elements upstream of the osteocalcin VDRE.
Evidence for a basal repressor in the 3' region of the human osteocalcin gene was obtained after cloning 3.5 kb of 3' flanking sequence into both OSCAT 2(OSCAT 7) and OSCAT 6(OSCAT8). The basal CAT activity of OSCAT 7 was 60% of that seen with the OSCAT 2 promoter. The basal CAT activity of OSCAT8 was similar to that of OSCAT2. Cloning this 3.5 kb of 3' sequence into heterologous promoters showed that the repressor function was promoter independent affecting the basal activity of all promoters tested. These studies indicate that regulatory elements exist in the human osteocalcin gene 5' and 3' of those elements already reported.
To test the hypothesis that these regions of the human osteocalcin gene were necessary to direct bone specific gene expression, the 3.8 kb 5' flanking sequence and the 3.5 kb 3' flanking sequence of the human osteocalcin gene were cloned together in correct orientation with unique restriction sites between them into which reporter and effector genes of interest could be cloned. This bone specific expression vector (pGOSCAS) has been tested with stable random integration into the mouse genome to direct bone specific gene expression of reporter genes. In a functional analysis of CAT activity pGOSCAS has been shown to direct bone specific CAT reporter gene expression with no ectopic expression of the reporter gene.
The pres

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