Application of multiple selections in gene targeting.

<p><b>A</b>. Genetic crosses of ends-out targeting based on the dual positive screening of <i>w+</i> and <i>Neo+</i> for targeting candidates, together with the negative selection of UAS-Rpr (Rpr+) for eliminating false positives. “X”: genotypes eliminated or greatly reduced in numbers by G418 selection or UAS-Rpr counter-selection. <b>B</b>. Map of pGX-attP-WN. pGX-attP-WN is a P-element based transforming vector. <i>5′P</i> and <i>3′P</i>: 5′ and 3′ P-element sequences; Amp^R: ampicillin-resistant gene.</p

Gene targeting of <i>Dscam-N</i> and <i>Dscam-C</i>.

<p><b>A</b>. Targeting design and PCR verification of <i>Dscam-N</i> and <i>Dscam-C</i> founder lines. Boxed are the genomic DNA (gDNA) structure and alternative-splicing patterns of <i>Dscam</i> locus. <i>Dscam</i> locus contains four alternative-splicing exons: 4, 6, 9 and 17 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#pone.0031997-Schmucker1" target="_blank">[13]</a>. Green boxes are gDNA regions used for 5′ and 3′ homology arms in the targeting constructs. In the <i>Dscam-N</i> founder knock-out line, a 5.7 kb genomic DNA covering the alternatively spliced exon 4 are deleted. In the <i>Dscam-C</i> founder knock-out line, a 7.6 kb genomic DNA covering the alternatively spliced exon 17 plus all the remaining downstream exons and 3′UTR are deleted. <i>Dscam-N</i> and <i>Dscam-C</i> founder knock-out lines carrying <i>W::Neo</i> marker are verified by 5′ and 3′ PCRs. 5′ or 3′ PCR is designed with one primer annealing within the W::Neo, while another primer anneals outside the gDNA region used for homology arms (“5′ gDNA” or “3′ gDNA”) in targeting constructs. Thus, only the correct targeting events will yield PCR products of expected size. <i>Dscam-N</i> and <i>Dscam-C</i> founder lines with <i>W::Neo</i> removed are further verified by dPCR-1 and dPCR-2. dPCR-1 is located within, while dPCR-2 spans over, the deleted region of <i>Dscam-N</i> or <i>Dscam-C</i>. <b>B</b>. 5′ and 3′ PCR-1 (red and yellow arrowheads, respectively) results from adults of <i>Dscam-N^GX07[w+]/CyO, Dscam-N^GX01[w+]/CyO, Dscam-C^GX101[w+]/CyO</i> and <i>Dscam-C^GX06[w+]/CyO</i>. <i>w¹¹¹⁸</i> was used as wild type control. White arrowheads pointing to non-specific PCR products. <b>C</b>. dPCR-1 (yellow arrowhead) and dPCR-2 (red arrowhead) results from embryos of <i>Dscam-N^GX01[w−]</i> and <i>Dscam-C^GX101[w−]</i> with <i>W::Neo</i> removed. <i>Dscam-N^GX01[w−]</i> and <i>Dscam-C^GX101[w−]</i> were balanced on <i>CyO, twi-GAL4, UAS-2xEGFP</i> (“CyO twiGFP”) chromosome so homozygous embryos could be distinguished by the absence of GFP. <i>w¹¹¹⁸</i> was used as the wild type control. For each PCR reaction genomic DNA was prepared by pooling approximately ten embryos together. For each sample, dPCR-1 and dPCR-2 reactions were carried out separately and were pooled before loading on the gel. MW: 1kb-plus DNA ladder (from Invitrogen); 5′ and 3′: the 5′ and 3′ homology arms of <i>Dscam-N</i> and <i>Dscam-C</i> targeting construct.</p

Generation of founder knock-out lines by ends-out targeting.

<p><b>^a.</b>Total estimated number of screening cross progeny screened in each targeting experiment. Because progeny of multiple vials or bottles were pooled and screened together, we did not register the clonality of the preliminary candidates. We assumed that each targeting mutant obtained was due to a distinct targeting event, based on the low HR frequency observed.</p><p><b>^b.</b>Since all female candidates were discarded in targeting experiments, the adjusted HR frequency should be twice higher than listed here.</p><p><b>^c.</b>Screening crosses were set up on the normal food first, then transferred to G418 food after two days.</p><p><b>^d.</b>A <i>dArf6^ΔKG#1</i> deletion allele generated by P-excision was used for complementation assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#pone.0031997-Huang4" target="_blank">[7]</a>.</p><p><b>^e.</b>Null allele of <i>P{PZ}Dscam⁰⁵⁵¹⁸</i> (BL#11412) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#pone.0031997-Schmucker1" target="_blank">[13]</a> was used for complementation assays.</p

Design of gene targeting for <i>dArf6</i>, <i>Dscam-N</i> and <i>Dscam-C</i> founder Knock-out lines.

<p>*: 5′+3′ Arms: the lengths of 5′ and 3′ homology arms in targeting construct.</p><p>**: According to <i>Drosophila</i> genome release FB2011.07 at <a href="http://www.flybase.org" target="_blank">www.flybase.org</a>.</p

Expression of <i>W::Neo</i> confers both <i>w+</i> and G418-resistance in flies.

<p><b>A</b>. Eye color in representative males from (clockwise from top right): <i>w¹¹¹⁸</i>, <i>y w; pArf6^GX22/TM3</i>, <i>y w; pDscam-N^GX113/TM3</i>, and <i>y w; pDscam-C^GX1/TM3</i>. <b>B</b>. G418-resistance of <i>W::Neo</i> transgenic donor lines and founder knock-out flies. Arrowheads indicate 0% survival rate. <i>FRT^[Neo]</i>: <i>y w; FRT-42D ubi::GFP^NLS/+</i>; <i>P{donor}</i>: transgenic donor insertion; <i>KO</i>: targeting allele; <i>w^[−]</i>: <i>TM3/+</i> and <i>CyO/+</i> cross progeny. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031997#s3" target="_blank">Materials and Methods</a> for detail genotypes.</p

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

<div><p>Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.</p> </div

Neuronal differentiation from iPSCs generated from fibroblasts.

<p>(a) Typical morphology of an iPS colony cultured for 7 days on matrigel with mTeSR1 medium. (b) Spherical cluster of cells containing neural stem/progenitor cells. (c) neural rosettes. (d) Neurosphere-like structures formed 1 day after culturing in suspension dissected spherical cluster and neural rosettes. (e) neural progenitor cells (NPCs). (f) neuron differentiating from neurosphere-like structures. (g–h) Staining of neurons with ß-tubulin III (Tuj1) (g) and MAP2 (h). Glutamate administration (10 µM) in NPCs cultures (i) did not evoked a significant calcium influx (Ca²⁺ basal levels: 0.26 ± 0.02; after glutamate: 0.27 ± 0.03; n = 128; not significant), whereas higher calcium basal levels were recorded in NPCs-neurons (j) (0.30 ± 0.02; n = 136; p<0.005 compared to NPCs calcium basal levels) and glutamate administration evoked a quicker and stronger response in term of Calcium influx (0.42 ± 0.03; n = 136; p<0.005 compared to calcium basal levels). The data are representative of four different experiments. The significant increase of glutamate-mediated Ca2+ influx suggests that the iPS-derived neurons are functional. Scale bar is 50 µm.</p

Mature neurons exposed to HCMV.

<p>Neuron-enriched cultures were infected with HCMV an MOI of 3. Expression of HCMV proteins was detected only in a small fraction of neurons (indicated by arrowhead) at day 3 post infection. (a) Bright-field. (b) Staining for early immediate, immediate and late HCMV antigens. (c) Nuclei counterstained with Hoechst. (d) Overlay of HCMV antigens and bright-field. (e–f) Staining for early immediate, immediate, and late HCMV antigens shows the presence of vial proteins (green) along neural processes at day 5 post infection. (f) Overlay of viral antigens and bright-field. Scale bar is 50 µm.</p

Microphotographs of HCMV-infected (a) and mock infected iPS cells.

<p>Scale bar is 50 µm.</p

Significant gene expression changes following HCMV infection of neural progenitor cells.

<p>List of genes significantly dysregulated in HCMV-infected neural progenitor cells 24 hours p.i. (p<0.05). FC: fold change; t-test p: p value for infected vs mock infected t-test. ‘X’s in squares indicate genes implicated in indicated categories of disorders at p<0.05. Neuronal-related genes are in bold. Ingenuity Pathways Analysis (IPA) results report p value ranges: Psychological disorders 1.62E-06-4.28E-02. Neurological Disease 1.26E-05-4.8E-02; Developmental Disorder 4.35E-05-2.7E-02; Infectious Disease 4.35E-05-3.23E-02.</p