A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

Abstract

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.

Humanization of bone and bone marrow in an orthotopic site reveals new potential therapeutic targets in osteosarcoma

“…Thus, we sought to evaluate its expression in patient samples and the humanized OS model. We found positive C12orf29 staining in the tumors, which was mainly extracellular as previously described [16], and a significantly upregulated gene expression in the tumors compared to the contralateral healthy femurs and non-tumor bearing ohTEBCs (Fig. 8E).…”

“…C12orf29 was expressed in a large number of patient tissues, although we did not find a co-expression with Col-II as expected due to previous reports (Fig. 9A-C) [16]. Using cell count analysis, we found a mean percentage of 24.1 ± 3.2% of C12orf29-positive cells when including all pre-chemotherapy patients.…”

“…We also demonstrate that human OS established within this humanized microenvironment utilizes the co-implanted human endothelial cells for tumor angiogenesis. Moreover, we show that the recently discovered gene C12orf29 [16] is being expressed in both our humanized OS model and in OS patients and may represent a potential new biomarker. The orthotopic humanized M A N U S C R I P T…”

“…Our group recently discovered the expression of C12orf29 in sheep bones and human fetal vertebras, and reported evidence for its role in skeletal maturation [16]. Thus, we sought to evaluate its expression in patient samples and the humanized OS model.…”

“…A previous study from our group led us to evaluate the expression of C12orf29 [16]. This gene is believed to promote the development of the musculoskeletal system; however, its cellular functions remain unknown.…”