CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line.

Abstract

The Swarm rat chondrosarcoma (RCS) cell lines derived from a spontaneous neoplasm in a rat spine several decades ago have provided excellent models of chondrosarcoma tumor development. In addition the robust chondrocyte phenotype (expression of a large panel of genes identical to that seen in normal rat cartilage) and the ability to generate cell clones have facilitated their extensive use in the identification of chondrocyte proteins and genes. The clustered regularly interspersed short palindromic repeat (CRISPR) technology employing the RNA-guided nuclease Cas9 has rapidly dominated the genome engineering field as a unique and powerful gene editing tool. We have generated a stable RCS cell line (RCS Cas9) expressing the nuclease Cas9 that enables the editing of any target gene or non-coding RNA by simple transfection with a guide RNA. As proof of principle, stable cell lines with targeted ablation of aggrecan expression (Acan KO) were generated and characterized. The studies show that stable chondrocyte cell lines with targeted genome editing can be quickly generated from RCS Cas9 cells using this system. The Acan KO cell lines also provided a tool for characterizing the response of chondrocytes to aggrecan loss and the role of aggrecan in chondrosarcoma development. Loss of aggrecan expression while not affecting the chondrocyte phenotype resulted in a much firmer attachment of cells to their substrate in culture. Large changes in the expression of several genes were observed in response to the absence of the proteoglycan matrix, including those for several small leucine rich proteoglycans (SLRPs), transcription factors and membrane transporters. Acan KO cells failed to form a substantial chondrosarcoma when injected subcutaneously in nude mice consistent with previous suggestions that the glycosaminoglycan-rich matrix surrounding the chondrosarcoma protects it from destruction by the host immune system. The studies provide new understanding of aggrecan function and the RCS Cas9 cell line is expected to provide a very valuable tool for the study gene function in chondrocytes.

• Authors: Maozhou Yang, Liang Zhang, Jeff Stevens, Gary Gibson
• Journal: Bone (Bone) Vol. 69 Pg. 118-25 (Dec 2014) ISSN: 1873-2763 [Electronic] United States
• PMID: 25260929 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Aggrecans
• Deoxyribonucleases

Topics

• Aggrecans (genetics)
• Animals
• Base Sequence
• Blotting, Western
• Cell Line
• Chondrocytes
• Clustered Regularly Interspaced Short Palindromic Repeats (genetics)
• Deoxyribonucleases
• Gene Knockout Techniques (methods)
• Mice
• Mice, Nude
• Molecular Sequence Data
• Phenotype
• Rats
• Rats, Sprague-Dawley
• Transcriptome
• Transfection