Bicistronic DNA construct comprising X-myc transgene for use in production of transgenic animal model systems for human hepatocellular carcinoma and transgenic animal model systems so produced

A transgenic mouse wherein the germ cells and somatic cells of the transgenic mouse comprise a bicistronic HBV X15-c-myc transgene, wherein said HBV X15-c-myc transgene comprises in sequence:

• a HBV X15 transgene and a c-myc transgene;
• wherein said bicistronic HBV X15-c-myc transgene encodes a truncated HBV X15 protein comprising amino acids 58-154 of HBV X15 and a murine c-myc protein, wherein said HBV X15 transgene comprises the nucleotide sequences disclosed in SEQ ID NO:1 and said murine c-myc transgene comprises exons 2 and 3 of murine c-myc;
• wherein said HBV X15 transgene is operatively linked to and under the regulatory control of its natural HBV X15 promoter and enhancer I elements and the c-myc transgene is operatively linked to and under the regulatory control of a core promoter and an enhancer II element of the HBV X15 gene;
• and
• wherein the expression of said HBV X15-c-myc transgene results in development of hepatocellular carcinoma in the transgenic mouse.

A method of screening a candidate substance to determine whether said candidate substance promotes hepatocellular carcinoma, wherein said method comprises the steps of:

• (i) providing a transgenic mouse wherein the germ cells and somatic cells of the transgenic mouse comprise a bicistronic HBV X15-c-myc transgene, wherein said HBV X15-c-myc transgene comprises in sequence:
• a HBV X15 transgene and a c-myc transgene;
• wherein said bicistronic HBV X15-c-myc transgene encodes a truncated HBV X15 protein comprising amino acids 58-154 of HBV X15 and a murine c-myc protein, wherein said HBV X15 transgene comprises the nucleotide sequences disclosed in SEQ ID NO:1 and said murine c-myc transgene comprises exons 2 and 3 of murine c-myc;
• wherein said HBV X15 transgene is operatively linked to and under the regulatory control of its natural HBV X15 promoter and enhancer I elements and the c-myc transgene is operatively linked to and under the regulatory control of a core promoter and an enhancer II element of the HBV X15 gene;
• and
• wherein the expression of said HBV X15 transgene results in the development of hepatocellular carcinoma in the transgenic mouse;
• (ii) exposing said transgenic mouse to said candidate substance;
• and
• (iii) monitoring said transgenic mouse for the development of hepatocellular carcinoma;
• (iv) wherein an increase in the development of hepatocellular carcinoma in the transgenic mouse exposed to said candidate substance compared to the development of the hepatocellular carcinoma in a transgenic mouse not exposed to said candidate substance indicates that the candidate substance promotes hepatocellular carcinoma.

A method of making a bicistronic HBV X15-c-myc transgene DNA construct comprising

• (a) subjecting a murine c-myc gene to a site directed mutagenesis to create an Nco I site near the first methionine codon of exon 2 thereof to produce a c-myc (2+3) gene comprising nucleotide sequences encoding exons 2 and 3 of c-myc;
• (b) joining a 539 base pair Dra I-Nco I fragment of the HBV genome encompassing the enhancer I and X promoter regions said c-myc (2+3) gene to generate Xp-c-myc gene;
• and
• (c) amplifying by polymerase chain reaction (PCR) a 362 base pair fragment of the HBV genome encompassing a coding region for the C-terminal half of the X gene, wherein the coding region encodes amino acids 58-154 of HBVX15, an enhancer II and a core promoter region, and cloning the 263 base pair fragment into the Nco I site of the Xp-c-myc construct to generate the HBV X15-c-myc transgene.