Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon.
OBJECTIVE
To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons.


METHODS
7 horses.


METHODS
Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB).


RESULTS
Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected byWLB in normal tendon and markedly increased in damaged tendons.


CONCLUSIONS
Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs.
Dahlgren
Linda A
LA
Comparative Orthopaedics Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USA.
Nixon
Alan J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006736:Horses; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid; D013710:Tendons / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger