Production of Recombinant RANKL for in vitro Osteoclastogenesis

Receptor Activator of Nuclear Factor _ B Ligand (RANKL) is a protein that belongs to the family of tumour necrosis factors (TNF) and is one of the two main inducers of osteoclast differentiation. It is therefore used in research facilities that study the molecular and cellular backgrounds of osteoarthritis (OA), a degenerative bone disease that is thought to be associated with a decline in osteoclast number or function. The aim of this work was to produce RANKL, using classical as well as novel methods and techniques of molecular biology and cell culture, including screening for the gene RANKL in leukaemic cell lines, amplification, transformation of Escherichia coli (E. coli) with a plasmid in which RANKL was previously inserted and subsequent analysis of clones, transfection of Chinese Hamster Ovary (CHO) cells with the purified plasmid, and finally qualitative analysis of the protein RANKL in the supernatant of a transfected CHO culture.

Autorentext

Silke Machauer graduated in Biotechnology (BSc) at Hamburg, University of Applied Sciences and shortly afterwards did her MSc in Pharmaceutical Biotechnology at the same University. After her graduation she started a PhD in Biochemistry/Molecular Biology at the University of Glasgow.

Klappentext

Receptor Activator of Nuclear Factor B Ligand (RANKL) is a protein that belongs to the family of tumour necrosis factors (TNF) and is one of the two main inducers of osteoclast differentiation. It is therefore used in research facilities that study the molecular and cellular backgrounds of osteoarthritis (OA), a degenerative bone disease that is thought to be associated with a decline in osteoclast number or function. The aim of this work was to produce RANKL, using classical as well as novel methods and techniques of molecular biology and cell culture, including screening for the gene RANKL in leukaemic cell lines, amplification, transformation of Escherichia coli (E. coli) with a plasmid in which RANKL was previously inserted and subsequent analysis of clones, transfection of Chinese Hamster Ovary (CHO) cells with the purified plasmid, and finally qualitative analysis of the protein RANKL in the supernatant of a transfected CHO culture.