Production of transgenic tilapia with Brockmann bodies secreting [desThrB30] human insulin.
BACKGROUND
Tilapia are commercially important tropical fish which, like many teleosts, have anatomically discrete islet organs called Brockmann bodies. When transplanted into diabetic nude mice, tilapia islets provide long-term normoglycemia and mammalian-like glucose tolerance profiles.


METHODS
Using site-directed mutagenesis and linker ligation we have "humanized" the tilapia insulin gene so that it codes for [desThrB30] human insulin while maintaining the tilapia regulatory sequences. Following microinjection into fertilized eggs, we screened DNA isolated from whole fry shortly after hatching by PCR. Positive fish were grown to sexual maturity and mated to wild-types and positive Fl's were further characterized.


RESULTS
Human insulin was detected in both serum and in the clusters of beta cells scattered throughout the Brockmann bodies. Surrounding non-beta cells as well as other tissues were negative indicating beta cell specific expression. Purification and sequencing of both A-and B-chains verified that the insulin was properly processed and humanized.


CONCLUSIONS
After extensive characterization, transgenic tilapia could become a suitable, inexpensive source of islet tissue that can be easily mass-produced for clinical islet xenotransplantation. Because tilapia islets are exceedingly resistant to hypoxia by mammalian standards, transgenic tilapia islets should be ideal for xenotransplantation using immunoisolation techniques.
Pohajdak
Bill
B
Department of Biology, Dalhousie University, Nova Scotia, Canada.
Mansour
Marc
M
Hrytsenko
Olga
O
Conlon
J Michael
JM
Dymond
L Clayton
LC
Wright
James R
JR
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015183:Restriction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013912:Threonine; D017210:Tilapia / Q000235:genetics*
D007328:Insulin; D009838:Oligodeoxyribonucleotides; D013912:Threonine