Cytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV) Sequences in the Pig Genome by Zinc Finger Nucleases (ZFN)

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors for transplantation and pigs are currently favoured as donor animals. However, xenotransplantation may be associated with the transmission of zoonotic microorganisms. Whereas most porcine microorganisms representing a risk for the human recipient may be eliminated by designated pathogen free breeding, multiple copies of porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated this way. PERVs are released as infectious particles and infect human cells. The zinc finger nuclease (ZFN) technology allows knocking out specifically cellular genes, however it was not yet used to eliminate multiple integrated proviral sequences with a strong conservation in the target sequence. To reduce the risk of horizontal PERV transmission and to knock out as many as possible proviruses, for the first time the powerful tool of the ZFN technology was used. ZFN were designed to bind specifically to sequences conserved in all known replication-competent proviruses. Expression and transport of the ZFN into the nucleus was shown by Western blot analysis, co- localisation analysis, PLA and FRET. Survival of transfected cells was analysed using fluorescent ZFN and cell counting. After transfection a strong expression of the ZFN proteins and a co-localisation of the expressed ZFN proteins were shown. However, expression of the ZFN was found to be extremely toxic for the transfected cells. The induced cytotoxicity was likely due to the specific cutting of the high copy number of the PERV proviruses, which is also commonly observed when ZFN with low specificity cleave numerous off- target sites in a genome. This is the first attempt to knock out multiple, nearly identical, genes in a cellular genome using ZFN. The attempt failed, and other strategies should be used to prevent PERV transmission

Easy and cost-effective stable positioning of suspension cells during live-cell imaging

Dynamic processes of cells can be best monitored when living cells are analyzed by imaging. While it is easy to observe adherent living cells it has been extremely challenging to analyze suspension cells. This cell type floats freely in the culture dish, and it is only a question of time when the focus or the observation field is lost. In order to keep the cells in focus, an easy and inexpensive method allowing the observation of living suspension cells during confocal laser scanning microscope imaging was developed

Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells—A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic

tANCHOR: A Novel Mammalian Cell Surface Peptide Display System

A novel tool for the presentation of peptides and small proteins on the surface of human cells has been developed. Our tANCHOR system utilizes tetraspanin anchors containing heterologous amino acid sequences inserted instead of the large extracellular loop. This technology allows a highly effective extracellular display of epitopes for antibody binding studies and many other potential applications.Peer Reviewe

Easy and cost-effective stable positioning of suspension cells during live-cell imaging

Dynamic processes of cells can be best monitored when living cells are analyzed by imaging. While it is easy to observe adherent living cells it has been extremely challenging to analyze suspension cells. This cell type floats freely in the culture dish, and it is only a question of time when the focus or the observation field is lost. In order to keep the cells in focus, an easy and inexpensive method allowing the observation of living suspension cells during confocal laser scanning microscope imaging was developed

The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

Human immunodeficiency virus type 1 (HIV-1) persists lifelong in infected individuals and has evolved unique strategies in order to evade the immune system. One of these strategies is the direct cell-to-cell spread of HIV-1. The formation of a virological synapse (VS) between donor and target cell is important for this process. Tetraspanins are cellular proteins that are actively involved in the formation of a VS. However, the molecular mechanisms of recruiting host proteins for the cell–cell transfer of particles to the VS remains unclear. Our study has mapped the binding site for the transmembrane envelope protein gp41 of HIV-1 within the large extracellular loop (LEL) of CD63 and showed that this interaction occurs predominantly at the VS between T cells where viral particles are transferred. Mutations within the highly conserved CCG motif of the tetraspanin superfamily abrogated recruiting of expressed HIV-1 GFP fused Gag core protein and CD63 to the VS. This demonstrates the biological significance of CD63 for enhanced formation of a VS. Since cell–cell spread of HIV-1 is a major route of persistent infection, these results highlight the central role of CD63 as a member of the tetraspanin superfamily during HIV-1 infection and pathogenesis

Localisation of the ZFN binding domains (set 1–3) in the sequence of PERV provirus.

<p>In addition, sequences of siRNA (gag2, pol1, pol2) successfully used for inhibition of PERV expression were indicated.</p

Expression of ZFN proteins in PK-15 cells.

<p><b>A.</b> Kinetic of expression of ZFN protein in PK-15 cells after nucleofection of different amounts (0.1 to 7.5 μg) of plasmids corresponding to ZFN set 1 (ZFN1, ZFN2, ZFN1/2). Both expressed proteins carry a 3xFLAG tag and for detection an antibody against the 3xFLAG tag was used. Cell lysate from PK-15 cells expressing an unrelated protein with a 3xFLAF tag and incubated for 2 days after nucleofection was used as positive control (Pos. ctrl). The arrows indicate the marker proteins. <b>B.</b> Detection of ZFN proteins in cytoplasmic and nuclear lysates. PK-15 cells were nucleofected with ZFN1 and ZFN2 plasmids together or separately and about 2 x 10⁶ cells per sample were fractionated. Each lane was loaded with nuclear or cytoplasmic extract from about 5 x 10⁵ cells. Whole cell lysate (WCL) from PK-15 cells transfected with pCMV, a vector with a FLAG tag was used as positive control (1 x 10⁵ cells). Anti-Flag antibodies were used for detection of the proteins. The purity of the cytoplasmic and nuclear fractions was analysed using antibodies against β-actin and DDX3, respectively.</p

Results of the surveyor nuclease assay.

<p><b>A.</b> PCR using genomic DNA as template for four different PCR (PCR1 = 645bp, PCR3 = 517 bp, PCR4 = 718 bp). After running on agarose gel, DNA concentration was estimated using ImageJ software. Lane 1–5, PK-15 cells transfected with 100 ng, 500 ng, 1 μg, 2 μg ZFN1/2 and 4 μg pLVTHM; lanes 8 and 12, PK-15 transfected with 2 μg ZFN1/2 each, lanes 9 and 13, PK-15 cells transfected with 4 μg pLVTHM. Lanes 6, 10 and 14 PERV-infected 293 cells transfected with 2 μg ZFN1/2 each and lanes 7, 11 and 15 with 4 μg pLVTHM. <b>B.</b> Agarose gel analysis of the rehybridisation of PCR amplicons shown in A. <b>C.</b> PAGE analysis of dehybridised and hybridised samples after incubation with 1 μl nuclease, 1 μl MgCl₂ and 1 μl enhancer for 20 minutes. The sample numbers correspond to the samples described in A. <b>D.</b> Analysis of the G/C control of the surveyor nuclease assay. Three different amounts (200, 400, 600 ng) of DNA were treated with nuclease as described by the manufacturer and analyzed on an agarose gel.</p

Primers used for the PCR.

<p>^aAccession nr. AJ293656</p><p>Primers used for the PCR.</p