Heparanase deficient non-human mammals
The invention relates to cells and transgenic non-human mammals having at least one disrupted heparanase allele. The invention further relates to methods of screening therapeutic drug candidates utilizing the heparanase deficient non-human mammals and cells.
1. A heparanase activity-eliminated transgenic mouse having a disrupted heparanase gene, wherein said disrupted heparanase gene lacks a HindIII-Xbal fragment comprising part of the promoter and exon1, and the transgenic mouse is homozygous for disruption of heparanse gene.
2. An isolated cell derived from a transgenic mouse according to claim 1 .
3. A method of making a transgenic mouse, comprising:
a) deleting part of the heparanase gene by homologous recombination in mouse embryonic stem cells, wherein the deleted part is the HindIII-Xbal fragment comprising part of the promoter and exon 1;
b) introducing recombinant cells obtained in step a) into an isolated blastocyst;
c) transplanting said blastocyst into a pseudopregnant mouse;
d) allowing said transplanted blastocyst to develop into a transgenic mouse;
e) breeding said transgenic mouse to produce offspring; and
f) screening said offspring to identify a heparanase activity-eliminated transgenic mouse.
4. The method according to claim 3 , wherein a nucleotide sequence of the deleted part in a) is depicted in SEQ ID NO: 1.
5. A vector for making a transgenic mouse according to claim 1 , said vector comprising a nucleic acid sequence encoding a heparanase knockout construct, wherein a part of the heparanase coding sequence is replaced with a selectable marker sequence, wherein said part is the HindIII-Xbal fragment comprising part of the promoter and exon 1.
6. The vector according to claim 5 , wherein said selectable marker sequence comprises a neomycin resistance gene.
7. A method of screening a therapeutic drug candidate, comprising
a) providing a transgenic mouse according to claim 1 ;
b) exposing said mouse to a disease stimulus
c) administering to said mouse said drug candidate; and
d) analyzing said mouse for development of a disease induced by said disease stimulus.
8. The method according to claim 7 , wherein said disease stimulus comprises inoculated tumor cells, and said step d) comprises determining formation of any tumor metastases.
9. The method according to claim 7 , wherein said disease stimulus is an inflammation stimulus, and said step d) comprises determining the level of any inflammatory responses.
10. The method according to claim 7 , wherein said disease stimulus induces experimental autoimmune encephalomyelitis.
11. The method according to claim 7 , wherein said disease stimulus induces an allergic reaction.
12. The method according to any one of claims 7 - 11 , further comprising comparing the results obtained to corresponding results obtained in a wild type mouse.