15 research outputs found
Isolation of cancer stem cells by selection for miR-302 expressing cells
Background Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more ‘differentiated’, i.e., less stem cell-like tumor cells. Methods Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. Results After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated. Conclusions We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells
The choroid plexus sodium-bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH
Constitutive transgene expression of Stem Cell Antigen-1 in the hair follicle alters the sensitivity to tumor formation and progression
The cell surface protein Stem Cell Antigen-1 (Sca-1) marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression
Design of the RMCE docking site.
<p>(A) <i>Left</i>: Schematics of the commercially available Flp-in docking site. <i>Right</i>: Design of the SB transposon constituting the RMCE docking site present in the new RMCE-in cell lines. The RMCE docking site contains the CAG promoter which drives the expression of a RFP reporter linked to the puromycin-resistant gene through the ribosomal skip element E2A. (B) Schematic representation of donor plasmid used for Flp-in (left) and RMCE-in (right). The genetic element of interest (GEI) is represented by a GFP reporter. Note that the GFP in the RMCE-in donor does not contain a poly(A)-signal and utilizes the poly(A) from the RMCE docking site. The RMCE-in and the Flp-in donor plasmid are compatible with both the RMCE-in and Flp-in cell line. (C) Post recombination of the Flp-in system <i>left</i>: Prokaryotic elements, the initial marker and selection gene are present in the commercial Flp-in<sup>™</sup>-293 cells post recombination, while the RMCE-in <i>right</i> leaves no prokaryotic DNA or initial reporter genes after cassette exchange.</p
Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System - Table 1
<p>Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System</p> - Table
Transposon copy number in the RMCE-in HEK, HeLa, and murine ES cell clones.
<p><b>(A)</b> Southern blot of selected genomic DNA from <i>KpnI</i> digested HEK, (<b>B)</b> HeLa and <i>PstI</i> digested (<b>C)</b> mES clones. A 700bp dCTP<sup>32</sup> labeled RFP probe (red rectangle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>) identified transposition of the gene cassette at bands > 2900bp as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>. Clone numbers are depicted above each lane.</p
Genomic mapping of chosen clones.
<p>Long-distance inverse PCR was used to map the position of the RMCE docking site integration in HEK colony 1.5 and 5.3, HeLa colony 2.7 and 3.6 <b>(A)</b>, and mES colony II-F1, II-H2, and I-F10 <b>(B).</b> Plasmid backbone (grey) and 5’ LIR sequences (purple) obtained by Sanger sequencing are shown above the area of insertion.</p
Validating flow cytometry results by fluorescence microscopy in RMCE-in HEK clones.
<p>HEK clones were exposed to 100X magnification and 1s exposure time for the assessment of RFP and GFP emission. All clones including colony 4.2 were RFP-positive and GFP with correlation to the high MFI value observed in flow cytometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g003" target="_blank">Fig 3A</a>).</p