gms | German Medical Science

Objectives: Within the last three years CRISPR/Cas9 (Crisp.) has become a powerful tool that allows manipulation of genomic DNA to generate isogenic cell lines for questions such as - but not exclusively - a.) multiplex knockout b.) promoter studies and c.) trafficking of molecules using reporter genes. For Crisp. clonal expansion of the engineered cells is necessary to give rise to a homozygous population after proper genetic analysis. This method is mainly limited to cell lines that have the potential to reconstitute themselves after single-cell suspension. In studies of bone metabolism, it is also of crucial importance to address questions of bone resorption - e.g. using osteoclast precursor cell lines such as RAW264.7 (RAW) - as well as bone production similarly. Widely used cell lines such as MC3T3 are limited in their ability for clonal expansion and do not share a BALB/c background with RAW osteoclast precursors. By using the K7M2 (K7) cell line, we want to introduce a bone cancer cell line, that shares the background of BALB/c, to allow comparable DNA sequences, as well as osteoclast/osteoblast co-culture assays. With this study, we asked I.) whether the K7 cell line shares the characteristics of osteoblasts including calcification and expression of well established marker genes of osteoblasts II.) whether the K7 cell line has the potential to reconstitute itself after single cell suspension and III.) whether knocking out a gene of interest in this cell line is successful to show general efficiency of the Crisp. method.

Methods: The K7 cell line was purchased from American Type Culture Collection (ATCC). Calcification of the cells was achieved by using osteogenic media supplemented with L-ascorbate and beta-glycerolphosphate that was changed every other day. Fixed cells were stained with Alizarin-Red to evaluate calcification on days 1,3,5,10,15,20,25,30 respectively. RNA was collected for qRT-PCR purpose to check the expression of various marker genes such as Alkaline Phosphatase (ALP), Collagen I (Col I) and Osteocalcin (OC). K7 cells were also stained for Osterix (OX) and RANKL using immunofluorescence. K7-GFP cells expressing the Green Fluorescence Gene (GFP) were transfected with plasmids hCas9 and MLM3636 against GFP. Successful targeting of the reporter gene was evaluated by fluorescence microscopy.

Results and Conclusion: CRISPR/Cas9 is a powerful tool for genomic engineering. With this study, we introduce the bone cancer cell line K7 as a suitable model to study genetically engineered bone cells. K7 represents hallmarks of osteoblasts such as expression of OX and RANKL confirmed by immunofluorescence, as well as calcification of the matrix under osteogenic conditions. Also the expression of osteoblastic marker genes (ALP, Col I and OC) represents characteristics of bone forming cells. Furthermore, successful knockout of GFP in K7-GFP cells provides evidence, that this cell line can serve as a model that allows approaches of genetic engineering to study bone cells in vitro.