A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep
| Autor: | Dietmar W. Hutmacher, Sally-Anne Stephenson, Yin Xiao, Jonathan P. Whitehead, Thor Friis |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
DNA
Complementary Blotting Western Biomedical Engineering Fluorescent Antibody Technique Medicine (miscellaneous) Bioengineering Mandible Biology Polymerase Chain Reaction Article Cell Line law.invention chemistry.chemical_compound Calcification Physiologic C12orf 29 law Complementary DNA pBluescript cDNA library Animals Humans Genomic library Cloning Molecular Gene Cells Cultured Polymerase chain reaction Gene Library Genetics Microscopy Confocal Osteoblasts Sheep pBluescript cDNA library PCR clone isolation method 060000 BIOLOGICAL SCIENCES chemistry Cell culture 090301 Biomaterials DNA |
| Zdroj: | Tissue Engineering Part C: Methods |
| ISSN: | 1937-3392 1937-3384 |
| DOI: | 10.1089/ten.tec.2013.0099 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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