PMID- 25260929
OWN - NLM
STAT- MEDLINE
DCOM- 20150731
LR  - 20141202
IS  - 1873-2763 (Electronic)
IS  - 1873-2763 (Linking)
VI  - 69
DP  - 2014 Dec
TI  - CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted 
      gene knockouts; analysis of an aggrecan knockout cell line.
PG  - 118-25
LID - S8756-3282(14)00335-4 [pii]
LID - 10.1016/j.bone.2014.09.005 [doi]
AB  - 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.
CI  - Copyright (c) 2014 Elsevier Inc. All rights reserved.
FAU - Yang, Maozhou
AU  - Yang M
AD  - Bone and Joint Center, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI, 
      USA. Electronic address: yang@bjc.hfh.edu.
FAU - Zhang, Liang
AU  - Zhang L
AD  - Bone and Joint Center, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI, 
      USA. Electronic address: liangz_tkl@yahoo.com.
FAU - Stevens, Jeff
AU  - Stevens J
AD  - Department of Internal Medicine, University of Iowa Carver College of Medicine, 
      Iowa City, IA, USA. Electronic address: jeff-stevens@uiowa.edu.
FAU - Gibson, Gary
AU  - Gibson G
AD  - Bone and Joint Center, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI, 
      USA. Electronic address: gibson@bjc.hfh.edu.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20140926
PL  - United States
TA  - Bone
JT  - Bone
JID - 8504048
RN  - 0 (Aggrecans)
RN  - EC 3.1.- (Deoxyribonucleases)
SB  - IM
MH  - Aggrecans/*genetics
MH  - Animals
MH  - Base Sequence
MH  - Blotting, Western
MH  - Cell Line
MH  - *Chondrocytes
MH  - Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
MH  - Deoxyribonucleases
MH  - Gene Knockout Techniques/*methods
MH  - Mice
MH  - Mice, Nude
MH  - Molecular Sequence Data
MH  - Phenotype
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Transcriptome
MH  - Transfection
OTO - NOTNLM
OT  - Aggrecan
OT  - CRISPR/Cas9
OT  - Chondrocyte
OT  - Chondrosarcoma
OT  - RNA guided gene modification
EDAT- 2014/09/28 06:00
MHDA- 2015/08/01 06:00
CRDT- 2014/09/28 06:00
PHST- 2014/08/05 00:00 [received]
PHST- 2014/09/05 00:00 [revised]
PHST- 2014/09/07 00:00 [accepted]
PHST- 2014/09/28 06:00 [entrez]
PHST- 2014/09/28 06:00 [pubmed]
PHST- 2015/08/01 06:00 [medline]
AID - S8756-3282(14)00335-4 [pii]
AID - 10.1016/j.bone.2014.09.005 [doi]
PST - ppublish
SO  - Bone. 2014 Dec;69:118-25. doi: 10.1016/j.bone.2014.09.005. Epub 2014 Sep 26.