Mutant library and targets () Localization of the area of the binding interface (bottom view) chosen for randomization (green) and interacting base pairs (−3, orange; −4, pink; −5, magenta)

<p><b>Copyright information:</b></p><p>Taken from " selection of engineered homing endonucleases using double-strand break induced homologous recombination"</p><p>Nucleic Acids Research 2005;33(20):e178-e178.</p><p>Published online 23 Nov 2005</p><p>PMCID:PMC1289081.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Zoom showing residues 44, 68 and 70 chosen for randomization (green), D75 (red) and interacting base pairs (−3, orange; −4, pink; −5, magenta). () Sequences of the target used for selection and screening. C1234, wild-type target C1221; and C4334, palindromic site derived from C1234. H1221 and H4334, palindromic sites related to C1221. Boxes highlight bases not found in sites C1221 or C4334

Impact of sdAb-Vpr1A on the G2-arrest and pro-apoptotic activities of Vpr.

<p>HeLa cells were co-transfected with plasmids for expression of GFP and HA-Vpr in combination with increasing concentrations of c-myc tagged sdAb Vpr1A expression plasmid when indicated. (<b>A, B</b>) <i>Cell cycle analysis</i>. 48 h after transfection, cells were fixed, permeabilized, and stained with propidium iodide. The DNA content was analyzed by flow cytometry on GFP-positive cells. In <b>A</b>, the cell DNA content profiles from a representative experiment are shown. The cells in G1, S and G2/M phases are indicated on the upper right panel. In <b>B</b>, results are expressed as the percentage of the G2M/G1 ratio relative to that measured in cells expressing HA-Vpr alone and are the means of 3 independent experiments. Error bars represent 1 S.D. from the mean. Statistical significance was determined using students <i>t</i> test (n.s., p>0.05; *, p<0.05). <b>C</b>) <i>Pro-apoptotic activity</i>. 72 h after transfection, cell surface PS exposure was analyzed by flow cytometry on GFP positive cells after staining with phycoerythrin-labelled Annexin V and 7AAD (7-Aminoactinomycin). Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure  =  relative to cells expressing HA-Vpr alone, and are the means of 3 independent experiments. Error bars represent 1 S.D. from the mean. Statistical significance was determined using students <i>t</i> test (n.s., p>0.05; *, p<0.05; **, p<0.01;***, p<0.001). <b>D</b>) <i>Expression of Vpr and sdAb Vpr1A proteins</i>. Lysates from HeLa transfected cells were analyzed by Western blotting using anti-HA (upper panel), anti-c-myc (middle panel) and anti-β-actin antibodies.</p

Selection system principle and sequences of selected binders.

<p><b>A)</b><i>Representation of the Sos Recruitment System principle</i>. The antigen (Ag) is fused to the human Sos protein (hSos) while sdAbs are fused to a myristoylation signal anchoring them in the plasma membrane. If the sdAb binds its Ag, hSos is driven to the membrane and activates the Ras pathway, leading to the growth of cdc25H mutant yeast at non-permissive temperature. <b>B)</b><i>Sequences of clones isolated by the SRS technique</i>. Two anti-Vpr and 2 anti-CA sdAbs were isolated by SRS. The sequences of their 3 complementary determining regions (CDRs) are shown. The frequency of each clone in the selection output (Freq.) is also reported.</p

Immunofluorescence assay using Vpr-GFP, HA-Vpr and sdAbs Vpr1A and Vpr2A.

<p>Immunofluorescence was performed by cotransfecting Hela cells with a plasmid coding for Vpr-GFP (<b>B</b>) or HA-Vpr (<b>C</b>) and increasing amount of a plasmid coding for a sdAb. Vpr-GFP, HA-Vpr and sdAbs were tracked as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113729#pone-0113729-g003" target="_blank"><b>Fig. 3</b></a>. The coefficient of Pearson (coefficient of co-localization) was calculated for each cell cotransfected with Vpr-GFP and a sdAb.</p

sdAb biochemical characterization.

<p><b>A</b>) <i>Purification of sdAbs produced into the cytoplasm of E. coli</i>. SDS-PAGE gel analysis was performed using comparable amount of sample for each step of the hexahistidine tag based purification process. Molecular weights (MW) are indicated. Soluble fraction of the total lysate (T), flow through (FT), washes (W) and elution fraction (E) were loaded in the presence or absence of β-mercaptoethanol (βME). Depicted results corresponding to sdAb Vpr1A are representative of what was obtained for other clones. <b>B</b>) <i>ELISA assay</i> w<i>ith purified sdAbs</i>. ELISA was performed by coating Maxisorp plates ON at 4°C with 10 µg/ml of GST-CA, GST-MA or Vpr. sdAbs were added at indicated concentrations (µg/mL). sdAb aCA2 was used as positive control (50 µg/ml) and the absence of sdAb was used as negative control.</p

Immunofluorescence assay using Gag-GFP, HA-CA and sdAbs CA7A and CA8B into HeLa cell.

<p>Immunofluorescence was performed by co-transfecting HeLa cells with a plasmid bearing HA-CA gene (<b>B</b>) or Gag-GFP gene (<b>C</b>) and a plasmid bearing a sdAb gene as indicated. Gag-GFP was tracked by GFP fluorescence. HA-CA was tracked using an anti-HA rat monoclonal antibody followed by an anti-rat monoclonal antibody coupled to AlexaFluor488 (green). SdAbs were detected using a mouse anti-c-myc monoclonal antibody followed by an anti-mouse antibody coupled to AlexaFluor555 (red). <b>Fig. 3A</b> are control cells transfected with the sdAb alone. <b>Fig. 3B</b> show cells transfected with HA-CA and <b>Fig. 3C</b> cells transfected with Gag-GFP. The coefficient of Pearson (coefficient of co-localization) was calculated for each co-transfected cell.</p

Drop assay after fresh transformation.

<p>Yeast co-transfected with either pSos-Nef, pSos-Vpr or pSos-CA or pSos (empty vector) and either pMyr-VHH19 (an anti-Nef sdAb previously described), pMyr-aCA2 (selected by phage display), pMyr (empty), pMyr-SB (Sos binding protein), or pMyr plasmids coding for Vpr1A, Vpr2A, CA7A or CA8B were resuspended and spotted onto galactose or glucose plates. Growth of colonies on galactose plates was indicated by + or - according with number and size of colonies.</p><p>Drop assay after fresh transformation.</p

Number of clones at each step of the selection process.

<p>Number of clones at each step of the selection process.</p

Highly Sensitive Single Domain Antibody–Quantum Dot Conjugates for Detection of HER2 Biomarker in Lung and Breast Cancer Cells

Despite the widespread availability of immunohistochemical and other methodologies for screening and early detection of lung and breast cancer biomarkers, diagnosis of the early stage of cancers can be difficult and prone to error. The identification and validation of early biomarkers specific to lung and breast cancers, which would permit the development of more sensitive methods for detection of early disease onset, is urgently needed. In this paper, ultra-small and bright nanoprobes based on quantum dots (QDs) conjugated to single domain anti-HER2 (human epidermal growth factor receptor 2) antibodies (sdAbs) were applied for immunolabeling of breast and lung cancer cell lines, and their performance was compared to that of anti-HER2 monoclonal antibodies conjugated to conventional organic dyes Alexa Fluor 488 and Alexa Fluor 568. The sdAbs–QD conjugates achieved superior staining in a panel of lung cancer cell lines with differential HER2 expression. This shows their outstanding potential for the development of more sensitive assays for early detection of cancer biomarkers