A Novel Method for Efficient Generation of Transfected Human Osteoclasts

Taylor, Adam; Rogers, Michael J.; Tosh, Denise; Coxon, Fraser P.

doi:10.1007/s00223-006-0245-6

Cited by 21 publications

(23 citation statements)

References 15 publications

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“…M-CSF-dependent macrophages were generated from healthy volunteers and transfected as previously described (69). Briefly, cultures of M-CSF-dependent macrophages were stimulated with 100 ng/ml recombinant human RANKL (rhRANKL; PeproTech) for 2 days prior to transfection.…”

Section: Methodsmentioning

confidence: 99%

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

et al. 2007

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This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

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Section: Methodsmentioning

confidence: 99%

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

et al. 2007

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“…32 Briefly, after cell harvesting, 1 Â 10 6 cells were pelleted and resuspended in human monocyte nucleofector solution (Lonza, Italy) to which 2 mg of plasmid were added. The transfected vectors were either empty or overexpressed the WT CLCN7 or the G215R CLCN7 mutation.…”

Section: Human Osteoclast Transfectionmentioning

confidence: 99%

“…32 We carried out four successful transfections and compared gene expression in the osteoclasts transfected with the mutated CLCN7 plasmid to the ones transfected with the WT CLCN7 plasmid. We observed, as expected, that the CLCN7 expression in osteoclasts transfected with WT CLCN7 vector was increased in comparison with those transfected with the empty vector (Figure 4a).…”

Section: Gene Expression Modifications In the Transfection Modelmentioning

confidence: 99%

Differentially expressed genes in autosomal dominant osteopetrosis type II osteoclasts reveal known and novel pathways for osteoclast biology

Coudert

Fattore

Baulard

et al. 2014

Laboratory Investigation

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Autosomal dominant osteopetrosis type II (ADO II) is a rare, heritable bone disorder characterized by a high bone mass and insufficient osteoclast activity. Mutations in the CLCN7 gene have been reported to cause ADO II. To gain novel insights into the pathways dysregulated in ADOII osteoclasts, we identified changes in gene expression in osteoclasts from patients with a heterozygous mutation of CLCN7. To do this, we carried out a transcriptomic study comparing gene expression in the osteoclasts of patients with ADO II and healthy donors. Our data show that, according to our selection criteria, 182 genes were differentially expressed in osteoclasts from patients and controls. From the 18 displaying the highest change in microarray, we confirmed differential expression for seven by qPCR. Although two of them have previously been found to be expressed in osteoclasts (ITGB5 and SERPINE2), the other five (CES1 (carboxyl esterase 1), UCHL1 (ubiquitin carboxy-terminal esterase L1, also known as ubiquitin thiolesterase), WARS (tryptophanyl-tRNA synthetase), GBP4 (guanylate-binding protein 4), and PRF1) are not yet known to have a role in this cell type. At the protein level, we confirmed elevated expression of ITGB5 and reduced expression of WARS, PRF1, and SERPINE2. Transfection of ClC-7 harboring the G215R mutation into osteoclasts resulted in an increased ITGB5 and reduced PRF1 expression of borderline significance. Finally, we observed that the ADO II patients presented a normal or increased serum level of bone formation markers, demonstrating a coupling between dysfunctional osteoclasts and osteoblasts. Sphingosine kinase 1 mRNA was expressed at the same level in ADO II and control osteoclasts. In conclusion, these data suggest that in addition to an acidification dysfunction caused by the CLCN7 mutation, a change in ITGB5, PRF1, WARS, and SERPINE2 expression could be part of the osteoclastic phenotype of ADO II.

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“…Nucleofection is an efficient nonviral transfection method for several difficult-to-transfect cell lines, such as primary neurons (Gartner et al, 2006), primary osteoclast cultures (Taylor et al, 2007) and human embryonic stem cells (Hohenstein et al, 2008). Differentiated OC-k3 cells were transfected using the Nucleofection technology from Amaxa (Cologne, Germany) as follows.…”

Section: Transient Transfection and Reporter Gene Assaymentioning

confidence: 99%