Molecular Characterization of Size and Copy Number of the Human Insulin Gene in Transfected Rat Islet Tumor Cell Clones

Summary

Two stable transfectants, NHI-6F and NHI-5B, obtained by cotransfection of the human insulin gene (a 15 kb EcoRI genomic fragment) with pSV2-neo into MSL-cells (pluripotent rat islet tumor cells), were characterized with regard to copy number and size of the inserted human sequences by Southern blot analyses using appropriate restriction enzymes. Digestion of total genomic DNA from the NHI-6F clone with EcoRI, Sacl and BgIII gave in all cases single human insulin gene fragments of 25 kb, 28 kb and 11 kb, respectively. From these data we were able to conclude that NHI-6F carries a single copy of the human insulin gene and that this copy has at least retained the 5 Bgll and the 3' Bglll flanking sites of human origin. Digestion of NHI-5B DNA gave a different banding pattern compared to NHI-6F (EcoRI: 20 kb; SacI: 7.8 kb; Bglll: 11 kb). These data indicate the presence of a single human insulin gene copy in clone NHI-5B with the preservation of the 5' SacI and 3' Bglll human flanking sites. In conclusion, the two transfected rat islet tumor clones, NHI-6F and -5B, carry a single copy of the human insulin gene. Each copy is presumably functionally active since known promoter and enhancer elements reside within 400 bp 5' to the gene. NHI-6F and -5B carry 2500 bp and 4500 bp 5' to the gene, respectively.

Key Words

Transfectants - Human insulin gene - Rat islet tumor cells - Copy number