Gene knockout in mouse embryonic stem cells.

<p>(<b>A</b>) Schematic of <i>EGFP</i> locus in BK4-G3.16 cells. The mouse ESC line BK4-G3.16 contains an <i>EGFP</i> gene stably integrated 5′ of the endogenous <i>Hprt</i> locus. E502, ZFN target site; hEF-1α, human elongation factor 1α promoter; pA, poly(A). The primer binding sites and the length (in base pairs) of the PCR amplicon are indicated. (<b>B, C</b>) Assessment of EGFP expression in BK4-G3.16 cells. Marker gene expression was determined by fluorescence microscopy (B) or flow cytometry (C) and was used to identify knockout clones (B) or to quantify gene disruption frequencies (C). (<b>D, E</b>) Dose-dependent gene disruption in mouse ESCs. BK4-G3.16 cells were lipofected (D) or electroporated (E) with increasing amounts of E502-specific ZFN expression vectors and an mCherry plasmid as an internal reference. The percentage of EGFP-negative cells was determined by flow cytometry at day 6 (D; n = 3; indicated is average and standard deviation) or at days 2, 6, and 8 (E; n = 1) post-transfection. (<b>F</b>) Kinetics of <i>EGFP</i> knockout. The graph displays the percentage of EGFP-negative cells (see D) from day 2 to day 9 post-transfection for two vector amounts (n = 3; indicated is average and standard deviation). WT, E502-WT; OH, E502-OH. (<b>G</b>) ZFN-induced mutations at E502 site. After lipofection of BK4-G3.16 with E502-OH expression vector, a genomic PCR fragment encompassing target site E502 was digested with the mismatch-sensitive T7 endonuclease I (T7E1). The expected lengths (in base pairs) of the T7E1 digestion products are shown on the right. Asterisks indicate an unspecific DNA fragment. Transfection with a vector control (Mock) was used as a control. (<b>H</b>) Expression kinetics. The graph displays the percentage of mCherry-positive BK4-G3.16 cells (see D) from day 2 to day 9 post-transfection.</p

Dataset for Antimicrobial resistance of Neisseria gonorrhoeae in Germany: low levels of cephalosporin resistance but rising azithromycin resistance

<p>This is Dataset used for Publication "Antimicrobial resistance of <em>Neisseria gonorrhoeae</em> in Germany: low levels of cephalosporin resistance but rising azithromycin resistance" and contains data on Information on performed Neisseria gonorrhoeae AMR tests in sentinel Laboratories as well as AMR testing in consiliary Laboratory.</p

Assessment of metaphase chromosomes and pluripotency.

<p>(<b>A–C</b>) Chromosome analysis. Metaphase spreads of BK4-G3.16 cells were stained with Giemsa. A representative picture of ∼20 analyzed metaphase spreads per clone is shown. (<b>D–L</b>) Assessment of pluripotency. ZFN-treated BK4-G3.16 cells were injected subcutaneously into immunodeficient mice and teratomas removed after 4–8 weeks. Histological analysis using hematoxylin/eosin staining revealed tissues derived from ectoderm (D, G, J), mesoderm (E, H, K) and endoderm (F, I, L). Scale bars = 100 µm for (D–G) and 50 µm for (H–L).</p

Gene knockout in U2OS.693 cells.

<p>(<b>A</b>) Schematic of ZFN-mediated knockout. A ZFN pair (ZFN_R and ZFN_L) designed to target position 502 in the <i>EGFP</i> gene (E502) creates a DNA double-strand break that is sealed by the error-prone non-homologous end-joining (NHEJ) pathway and hence leads to disruption of the coding sequence. (<b>B</b>) Dose-dependent gene disruption in U2OS.693 cells. U2OS.693 cells that stably express a destabilized EGFP were transfected with increasing amounts of E502-specific ZFN expression vectors (75–1200 ng). The percentage of EGFP-negative cells was determined 6 days post-transfection by flow cytometry (n = 3; indicated is average and standard deviation). E502-WT, ZFN with wild-type FokI domain; E502-OH, ZFN with obligate heterodimeric FokI domain. (<b>C</b>) ZFN expression levels. After co-transfection of HEK293T cells with ZFN expression vectors and pEGFP, cell lysates were probed with antibodies against the HA tag and EGFP. Amount of transfected ZFN plasmids was 75 ng, 300 ng, and 1200 ng. NT, non-transfected cells. (<b>D</b>) Kinetics of <i>EGFP</i> knockout. The graph displays the percentage of EGFP-negative cells (see B) from day 1 to day 6 post-transfection for two vector amounts (n = 3; indicated is average and standard deviation). WT, E502-WT; OH, E502-OH.</p

Molecular characterization of individual ESC clones.

<p>(<b>A</b>) Schematic of <i>EGFP</i> locus in BK4-G3.16 cells. The primer binding sites and the lengths (in base pairs) of the generated PCR amplicons are indicated. (<b>B</b>) Genotyping. The genomic PCR amplicon encompassing target site E502 was digested with TaqI. The positions of the TaqI-resistant DNA fragment (228 bp) and a cleavage product (132 bp) are indicated. (<b>C, D</b>) Sequence of disrupted <i>EGFP</i> alleles. A genomic PCR expected to produce a 556-bp amplicon was evaluated by agarose gel electrophoresis (C) and the isolated DNA fragments sequenced (D). The positions of the expected (556 bp) and the additional DNA fragment (415 bp) are indicated in (C), the sequences of all alleles are shown in (D). The ZFN target site (subsites E502L and E502R) is highlighted in capital letters, the TaqI site in bold letters.</p