Efficient and stable gene expression into human osteoclasts using an HIV-1-based lentiviral vector.
Since osteoclasts are terminally differentiated cells without proliferating activity, efficient and stable gene expression into these cells remains a difficulty. In the current study, we investigate gene transduction into human preosteoclasts by a replication defective lentivirus-based vector containing a modified HIV-1 genome. Human preosteoclasts (differentiating osteoclasts) were transduced with lentiviruses bearing an enhanced green fluorescent protein (EFGP) reporter gene. Transduction efficiencies were measured by flow cytometry for EGFP protein expression. Sorted human transduced preosteoclasts were replated and differentiated under human macrophage colony-stimulating factor and human receptor activator of NF-kappaB ligand. Mature osteoclasts were then analyzed by the cell viability assay, TRACP assay, and pit formation assay. Efficient gene transduction was obtained at multiplicity of infection of 10, and gene expression lasted for over 4 weeks using our protocol. Lentiviral transduction did not affect osteoclast survival, formation, or function. These results establish an efficient method for gene transduction into human preosteoclasts using a lentiviral vector. Importantly, these transduced preosteoclasts could differentiate into mature osteoclasts without a negative impact from the lentiviruses. This protocol provides a new tool for studies of osteoclast biology. Further work in this area may open new avenues for the study of osteoclast gene signaling and gene therapy of disorders of osteoclast function.
Chu
Kang
K
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. kachu@iupui.edu
Cornetta
Kenneth G
KG
Econs
Michael J
MJ
D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism* / Q000821:virology*; D014161:Transduction, Genetic
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins