A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep.
The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.
1 Regenerative Medicine Group, Institute of Health and Biomedical Innovation, Queensland University of Technology , Brisbane, Queensland, Australia .
Thor Einar
TE
Friis
Sally
S
Stephenson
Yin
Y
Xiao
Jon
J
Whitehead
Dietmar W
DW
Hutmacher
D000818:Animals; D015153:Blotting, Western; D002113:Calcification, Physiologic; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D006801:Humans; D008334:Mandible / Q000166:cytology; D018613:Microscopy, Confocal; D010006:Osteoblasts / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012756:Sheep / Q000235:genetics*
D018076:DNA, Complementary