Part:BBa_K415500

BMP2 L1L2 MammoBlock Entry Vector

BBa_K415500 is an L1L2 Entry Vector for MammoBlock recombination cloning, containing the gene BMP2 (see below)

BMP2 (Bone Morphogenetic Protein 2) is a well estabished pro-osteogenic morphogen shown to induce osteogenesis in a variety of cell types in literature. Upon binding BMP2, the BMPRI receptor activates a downstream signaling cascade involving Smad proteins, which results in activation of the Runt-related transcription factor 2 (RUNX2). RUNX2 is the main transcription factor controlling osteoblast differentiation; transfection of RUNX2 induced osteoblastic differentiation of fibroblasts, which do not normally differentiate into osteoblasts.

Characterization

Figure 1. Morphology Results Summary.

Morphology Following BMP2 Stimulation

Human Recombinant BMP2 with sequence identical as encoded by K415500 was harvested from E. Coli from Insight Genomics, VA Reconstituted in 20 mM acetic acid in H2O to 100 ug/mL, total 90 uL. BMP2 protein and added it to the supernatant of stem cell cultures at 100ng/ml and 300ng/ml, based on a review of concentrations used in the literature. We tested the response to both constitutive (all 5 days) and transient (induction stopped on day 2) BMP2 signaling. HEK cells were also tested as a negative control for differentiation. Micrographs were taken on day 5 to assay for changes morphology.

Results

Both C2C12 and C3HT101/2 were affected by the addition of BMP2 (Figure 1). C3HT101/2 exhibited the strongest response; at 300ng/ml of BMP2, we observed small mineralized nodules beginning to form, as previously described in the literature. However there was negligible change in morphology of the transiently stimulated C3HT101/2 cells, indicating that BMP2 induction is required over a longer time period to induce differentiation. The C2C12 uninduced cells began differentiation into muscle tissue, seen in multinucelated myofibril formation; the C2C12 cells induced with BMP2 instead began to exhibit morphological osteoblast markers. We observed no significant changes in HEK cell morphology.

Figure 2. Photograph of ALP Assay Results.

ALP Assay

Alkaline Phosphatase (ALP) assay was performed on on the cultures above. The ALP assay is a routine protocol used to determine differentiation. It measures activity of an osteoblast-specific protein, alkaline phosphatase. This enzyme increases the local concentration of phosphate, which aids formation of the the hydroxyapatite ion that underlies mineralization in bone.

Results

C2C12 displayed a strong increase in alkaline phosphatase activity in cells treated with 100ng/ml and 300ng/ml BMP2, when compared with untreated controls (Figure 2). The C3HT101/2 mesenchymal stem cells exhibited a significantly weaker, but still visible upregulation of ALP in cells treated with BMP2. HEK and transiently stimulated C3HT101/2 did not display visible upregulation of ALP activity.

Characterization of BMP2's effects in cartilage regeneration (by KEYSTONE_A 2020)

BMP2 and VEGF inhibition promote expression of cartilaginous pathways on sorted skeletal stem-cell (SSC) in vitro (Figure 3). Skeletal stem-cells (SSCs) isolated from the articular surface, periosteum and bone marrow all produced higher amounts of cartilage with addition of BMP2 + sVEGFR1 when compared with addition of PBS only (Figure 4). For in vivo experiments, cotransplantation with BMP2 + sVEGFR1 can also promote cartilage formation (Figure 5).

Reference

Murphy, M. P., Koepke, L. S., Lopez, M. T., Tong, X., Ambrosi, T. H., Gulati, G. S., ... & Steininger, H. (2020). Articular cartilage regeneration by activated skeletal stem cells. Nature Medicine, 1-10.

Figure 3. SSC gene expression levels after in vitro treatment of PBS, BMP2, or sVEGFR1.

Figure 4. Alcian blue staining (for acidic glycans in cartilage) of SSC following chondrogenic differentiation with treatment of PBS or BMP2 + sVEGFR1

Figure 5. Cotransplantation with BMP2 + sVEGFR1 led to increased cartilage formation in vivo

-=Sequence and Features=-

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1218
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1218
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1218
  Illegal BamHI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1218
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1218
  Illegal AgeI site found at 11
  Illegal AgeI site found at 313
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 218