The EC4 European syllabus for post-graduate training in clinical chemistry and laboratory medicine : Version 4-2012

Laboratory medicine’s practitioners across the European community include medical, scientific and pharmacy trained specialists whose contributions to health and healthcare is in the application of diagnostic tests for screening and early detection of disease, differential diagnosis, monitoring, management and treatment of patients, and their prognostic assessment. In submitting a revised common syllabus for post-graduate education and training across the 27 member states an expectation is set for harmonised, high quality, safe practice. In this regard an extended ‘Core knowledge, skills and competencies’ division embracing all laboratory medicine disciplines is described. For the first time the syllabus identifies the competencies required to meet clinical leadership demands for defining, directing and assuring the efficiency and effectiveness of laboratory services as well as expectations in translating knowledge and skills into ability to practice. In a ‘Specialist knowledge’ division, the expectations from the individual disciplines of Clinical Chemistry/Immunology, Haematology/Blood Transfusion, Microbiology/ Virology, Genetics and In Vitro Fertilisation are described. Beyond providing a common platform of knowledge, skills and competency, the syllabus supports the aims of the European Commission in providing safeguards to increasing professional mobility across European borders at a time when demand for highly qualified professionals is increasing and the labour force is declining. It continues to act as a guide for the formulation of national programmes supplemented by the needs of individual country priorities.peer-reviewe

Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes)

Transduction of target cells by CsCl density gradient-purified dHV.F50 and dHV.68/5′3′.F50 particles.

<p>(A) Direct fluorescence microscopy analysis of HeLa cells and DMD myoblasts that were either mock-infected or infected with 4, 20 or 100 HTUs of dHV.F50 or dHV.68/5′3′.F50 per cell. (B) Representative flow cytometry histograms corresponding to HeLa cells and DMD myoblasts transduced with different amounts of dHV.F50 or dHV.68/5′3′.F50 (i.e. 0, 4, 20 or 100 HTUs/cell). The % of maximum refers to the ratio between the number of events analyzed in each case and the number of events in the sample with the largest number of events. (C) Quantification by flow cytometry of gene transfer into HeLa cells and DMD myoblasts exposed to 4, 20 or 100 HTUs of dHV.F50 or dHV.68/5′3′.F50 per cell (<i>n</i> = 3). Gene transfer activity is expressed both in terms of the frequency of eGFP-positive cells and the mean fluorescent intensity (MFI) of the transduced cells (which reflects mean intracellular eGFP levels). Error bars represent standard deviations. All measurements were performed at 72 hours postinfection.</p

Propagation of <i>rep68</i>-positive and fiber-modified dHVs.

<p>(A) Direct fluorescence microscopy analysis of the PER.tTA.Cre76 cells used for the serial propagation of dHV.F50, dHV.68/5′3′.F50 and dHV.68/3′5′.F50. (B) Flow cytometric analysis of HeLa cells employed for the determination of the <i>eGFP</i> transfer activity of clarified producer cell lysates derived from consecutive rounds of vector amplification. P1, passage 1; P2, passage 2; P3, passage 3; P4, passage 4.</p

Examination of targeted versus random dHV.68/5′3′.F50 DNA insertion through Southern blot analysis of genomic DNA.

<p>(A) Upper panel, Eco32I restriction map of the AAV DNA preintegration region (i.e. the <i>AAVS1</i> locus) on human chromosome 19 at 19q13.3-qter. Vertical line and solid oval, trs and RBE, respectively. The vertical solid arrows indicate Eco32I recognition sites delimiting a 5.6-kb DNA segment. The solid horizontal bar denotes the <i>AAVS1</i>-specific probe drawn in relation to its target sequence (diagram not on scale). Lower panel, Eco32I restriction map of dHV.68/5′3′.F50 DNA showing the various hybridization probes (horizontal solid bars) drawn in relation to their cognate target sequences (drawing not on scale). For an explanation of symbols and abbreviations, see the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003084#pone-0003084-g001" target="_blank">Fig. 1</a>. (B) Ten micrograms of total cellular DNA from 10 representative clones derived from HeLa cells co-infected with dHV.68/5′3′.F50 and hcAd.FLPe.F50 were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA fragments were subjected to Southern blot analysis with the <i>AAVS1</i>-specific probe (<i>AAVS1</i> probe; upper panel). After removal of the <i>AAVS1</i>-specific probe, the transferred DNA was incubated simultaneously with a probe derived from the AAV p5IEE plus the <i>rβG</i> gene pA signal and a probe specific for the <i>eGFP</i> ORF (5′ and 3′ vector probes; middle panel). Finally, following a second stripping step, the transferred DNA was exposed to a fourth probe corresponding to the 1.9-kb Eco32I fragment in the dHV.68/5′3′.F50 genome (internal vector probe; lower panel). Clones derived from HeLa cells with AAV Rep68-induced disruptions in the <i>AAVS1</i> locus are indicated by solid arrows, whereas those that also have dHV.68/5′3′.F50 DNA inserted into this specific region of human chromosome 19 are discriminated by open arrows. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (C) Ten micrograms of total cellular DNA from 10 clones of DMD myoblasts co-infected with dHV.68/5′3′.F50 and hcAd.FLPe.F50 were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA species were subjected to Southern blot analysis with the <i>AAVS1</i>-specific probe (<i>AAVS1</i> probe; upper panel) and, after its removal, with the two probes binding close to the termini of the dHV.68/5′3′.F50 genome (5′ and 3′ vector-specific probes; lower panel). Clones derived from DMD myoblasts with AAV Rep68-induced disruptions in the <i>AAVS1</i> locus are indicated by solid arrows, whereas those that also have dHV.68/5′3′.F50 DNA inserted into this specific region of human chromosome 19 are discriminated by open arrows. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (D) Ten micrograms of total cellular DNA from 9 representative clones derived from HeLa cells infected with dHV.68/5′3′.F50 alone were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA fragments were subjected to Southern blot analysis with the <i>AAVS1</i>-specific probe. Marker, GeneRuler DNA Ladder Mix molecular weight marker.</p

Frequencies of targeted foreign DNA integration events derived from Southern blot analysis of dHV.68/5′3′.F50-transduced HeLa cell and DMD myoblast clones.

<p>Frequencies of targeted foreign DNA integration events derived from Southern blot analysis of dHV.68/5′3′.F50-transduced HeLa cell and DMD myoblast clones.</p

Testing the inducible <i>rep68</i> expression unit in dHV.68/5′3′.F50 particles purified by CsCl density gradient ultracentrifugation.

<p>(A) Western blot analysis of FLPe-induced Rep68 synthesis. HeLa cells were co-transduced with dHV.F50 and hcAd.FLPe.F50 (lane 1) or with dHV.68/5′3′.F50 and hcAd.FLPe.F50 (lane 3) or were transduced exclusively with dHV.F50 (lane 2) or with dHV.68/5′3′.F50 (lane 4). Three days postinfection, cellular proteins were extracted, separated in an SDS-10% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Next, the membrane was incubated with a monoclonal antibody specific for all four AAV <i>rep</i> gene products. An antibody directed against actin served as a loading control. (B) eGFP direct fluorescence microscopy and Rep68 immunofluorescence microscopy on HeLa cells infected with hcAd.FLPe.F50 (50 GSAUs/cell) and either dHV.F50 (5 HTUs/cell; left column) or dHV.68/5′3′.F50 (5 HTUs/cell; right column). The pictures are derived from representative microscopic fields. The fluorescent signals derived from the green and red channels are overlaid in the lower panels. The arrowheads in the right panels point to cells containing both eGFP and Rep68. Note that the former protein is distributed throughout the cell whereas the latter is confined to the nucleus. Original magnification: 400×.</p

Testing the functionality of pGS.pA+.Rep68 and hcAd.FLPe.F50.

<p>Extrachromosomal DNA was extracted from PER.tTA.Cre76 cells co-transfected with the rAAV vector shuttle plasmid pAAV.DsRed and the FLP-activatable <i>rep68</i> expression plasmid pGS.pA+.Rep68 and infected 24 h later with Ad.floxedΨ.F50 alone (lane 1) or with Ad.floxedΨ.F50 plus hcAd.FLPe.F50 (lane 2). Positive (lane 3) and negative (lane 4) controls consisted of extrachromosomal DNA extracted from Ad.floxedΨ.F50- and mock-infected PER.tTA.Cre76 cells, respectively, co-transfected with pAAV.DsRed and the constitutive <i>rep78/68</i> expression plasmid pGAPDH.Rep78/68. The extracts were treated with DpnI to selectively digest input, non-replicated, prokaryotic DNA and were subjected to Southern blot analysis using a probe corresponding to the <i>DsRed.T4</i> ORF. Lane M, GeneRuler DNA Ladder Mix molecular weight marker (Fermentas). The positions and sizes (in kb) of the rAAV replicative intermediates (i.e., DMs and DDs) are indicated. The numerals at the left correspond to restriction DNA fragment sizes in kb.</p

Integration of exogenous DNA into <i>AAVS1</i> after gene transfer with <i>rep68</i>-positive dHV.68/5′3′.F50 particles.

<p>(A) Overview of the PCR-based targeted DNA integration assay. Genomic DNA extracted from HeLa cell clone N+2 and DMD myoblast clone 3C5 served as control templates derived from cells genetically modified through vector DNA insertion outside and inside the <i>AAVS1</i> locus, respectively. The experimental samples consisted of chromosomal DNA from DMD myoblasts infected with dHV.68/5′3′.F50 alone (40 HTUs/cell) or co-infected with dHV.68/5′3′.F50 (40 HTUs/cell) and hcAd.FLPe.F50 (40 GSAUs/cell). The upper part of the boxed pictogram represents the section of human chromosome 19 at 19q13.3-qter harboring the <i>AAVS1</i> locus. Vertical line and adjacent oval, trs and RBE, respectively; horizontal fading bar, AAV DNA preintegration region; green box, representation of a targeted vector DNA insertion event leading to an <i>AAVS1</i>-foreign DNA junction; numbered black and green arrows symbolize <i>AAVS1</i>- and vector DNA-specific primers, respectively. (B) Agarose gel electrophoresis of PCR products resulting from amplifications carried out on chromosomal DNA from HeLa cell clone N+2 (lanes 1), DMD myoblast clone 3C5 (lanes 2), DMD myoblast population transduced with dHV.68/5′3′.F50 alone (lanes 3) and DMD myoblast population co-transduced with dHV.68/5′3′.F50 and hcAd.FLPe.F50 (lanes 4). Lanes M, Gene Ruler DNA Ladder Mix molecular weight marker. Upper panel, internal control PCR products resulting from amplifications performed with the <i>AAVS1</i>-specific primers # 211 and # 196. Lower panel, nested PCR products of amplifications carried out with oligodeoxyribonucleotides # 212 and # 188 on DNA synthesized with the aid of primers # 211 and # 651. (C) Nucleotide sequence analysis of junctions generated between <i>AAVS1</i> and exogenous DNA following co-transduction of DMD myoblasts with dHV.68/5′3′.F50 and hcAd.FLPe.F50. The DNA sequencing data in the upper and lower panel correspond to inserts derived from PCR amplifications on chromosomal DNA from myoblast clone 3C5 and from the DMD myoblast population co-transduced with dHV.68/5′3′.F50, respectively. The black and green bars indicate <i>AAVS1</i> and vector DNA, respectively. Vertical arrows points to the junction between <i>AAVS1</i> and dHV.68/5′3′.F50 DNA present in each individual clone.</p