Plasmid Electroporation Research Articles

Mechanisms and molecular typing of carbapenem-resistant Proteus mirabilis strains deficient in swarming motility

Objective To investigate the mechanism and epidemiological characteristics of carbapenem-resistant Proteus mirabilis (PM) strains deficient in swarming motility. Methods PM strains were isolated from Hangzhou General Hospital of CAPF (Chinese People′s Armed Police Forces) during January 2013 to December 2014. Bacterial motility and flagella of the PM strains were observed through semi-solid agar culture and flagella staining. Pulsed-field gel electrophoresis (PFGE) was performed for homology analysis. Antimicrobial susceptibility test and phenotypic confirmatory test were also carried out. PCR analysis and DNA sequencing were performed to confirm the genotype of resistant genes. Plasmid electroporation and S1-PFGE in combination with Southern blot hybridization were used to determine the location of the carbapenem-resistant genes. Genetic structure of the blaKPC-2 gene was obtained by PCR mapping. Results A total of 42 PM isolates deficient in swarming motility were screened out and the resistance rates to imipenem and meropenem were 57.1% and 52.4%, respectively. PCR analysis and DNA sequencing confirmed that 24 carbapenem-resistant PM isolates deficient in swarming motility carried blaKPC-2 gene and belonged to three clones as indicated by the results of PFGE. Southern blot hybridization indicated that the blaKPC-2 gene was located on plasmids varying in size (26 kb, 55 kb and 139 kb). In addition, some of the strains harbored several resistant genes, such as blaTEM-1, blaCTX-M-65 and rmtB. The genetic structures of strains carrying blaKPC-2 gene were ISKpn8, blaKPC-2 and ISKpn6-like from upstream to downstream. Conclusion Compared with the PM strains with swarming motility, the carbapenem-resistance rate was significantly higher in these PM strains deficient in swarming motility. Carbapenemases KPC-2 played an important role in the carbapenem-resistant PM strains deficient in swarming motility. There was a cloning spread trend for carbapenem-resistant PM strains in our hospital. Clinicians should pay more attention to the risk of spreading. Key words: Proteus mirabilis; Swarming motility; Carbapenemase; Flagellum; Nosocomial infection

Construction of Novel Plasmid Vectors for Gene Knockout in Helicobacter pylori.

Helicobacter pylori infection plays an important role in the etiology of various gastroduodenal diseases. However, pathogenic mechanism of H. pylori is not clear. More potential pathogenic factors need to be further discovered and studied. In this study, two vectors for generating double-crossover recombination gene knockout plasmids in H. pylori were designed based on the backbone of plasmid pLYL03. Genes on plasmid pLYL03 were rearranged, and the redundant sequences were reduced. Erythromycin-resistant gene on pLYL03 was replaced by aphA or catGC to generate the kanamycin-resistant plasmid pSJHK or the chloramphenicol-resistant plasmid pSJHC. The sizes of pSJHK and pSJHC are 4371 and 3949 bp, respectively. Based on plasmids pSJHK and pSJHC, double-crossover recombination gene knockout plasmids targeting hp0169 and hp0788 were constructed, and deletion mutants were achieved by electroporation of the gene-targeting plasmids. The results indicated that plasmids pSJHK and pSJHC are efficient for gene deletion in H. pylori, and the transformation efficiency of pSJHC is slightly higher than pSJHK. These plasmids provide convenient genetic tools for further research of novel pathogenic factors in H. pylori. Derivative plasmids developed by changing antibiotic-resistant genes would also provide valuable tools for the study of functional genes in other bacteria.

Abstract 4884: Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses

Abstract Interleukin-12 (IL-12), produced by antigen-presenting cells, plays a pivotal role in the interplay between innate and adaptive arms of the immune system. IL-12 treatment has been shown to augment cytotoxic T lymphocyte (CTL) and T-helper 1 responses and anti-tumor effects. However, its use as a systemic therapeutic agent is limited due to toxicity. Intratumoral administration of IL-12 is thus being explored as a local alternative route of administration. Such strategies involve plasmid electroporation or other methods that randomly direct cells in the tumor to express IL-12. Here, we evaluated whether targeting expression of IL-12 to intratumoral dendritic cells (DC), thus mimicking the cytokine's physiological biosynthesis and localization, would result in local and systemic immune responses and tumor control in preclinical models. Six murine tumor models were used, including melanoma (B16F10), colon carcinoma (CT26), breast cancer (4T1), lymphoma (A20) and mastocytoma (P815). Tumors were implanted unilaterally, and for some models also bilaterally, to study systemic (abscopal) effects of therapy. A chimeric third-generation lentiviral vector from the ZVex platform, pseudotyped with the DC-tropic envelope glycoprotein of Sindbis virus, was engineered to express murine IL-12 (p35-p40) (ZVex/IL-12). ZVex/IL-12 was administered as a single intratumoral injection into palpable tumors, alone or in combination with the synthetic TLR4-agonist, glucopyranosyl lipid A (GLA). In some models, systemic anti-CTLA4 treatment was added to enhance clinical efficacy. Animals were monitored 2-3 times per week for tumor size and survival. Greatest curative efficacy of ZVex/IL-12 was observed in the 100% lethal CT26 flank model, where all treated animals cleared their tumors and survived until end-of-study at 70 days post-challenge. In the B16 footpad and flank models, curative efficacy varied from 40% to 90%, with abscopal effects being observed after addition of anti-CTLA4. In the A20 and P815 models, efficacy varied from 30% to 60%. In the aggressive orthotopic 4T1 breast cancer model, co-administration of ZVex/IL-12 with GLA-AF resulted in significantly delayed tumor growth and increased survival time compared to either ZVex/IL-12 or GLA-AF used alone. A single intratumoral injection of ZVex/IL-12 resulted in complete tumor regression or significant growth delay in all mouse tumor models investigated and was accompanied by significant survival benefit. The therapeutic effect could be enhanced by ad-mixing with GLA. Abscopal effects were observed in some models after treatment with ZVex/IL12 only and in others after addition of anti-CTLA4. These results point to the powerful modification of the tumor/tumor microenvironment by intratumorally expressed IL-12 by a ZVex vector and the induction of local and systemic immunity. Citation Format: Tina C. Albershardt, Anshika Bajaj, Jacob F. Archer, Rebecca S. Reeves, Andrea J. Parsons, Lisa Y. Ngo, Jan ter Meulen, Peter Berglund. Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4884.

Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.

Adult neural stem cells (aNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCs in their intact niche and during regeneration in vivo should facilitate the identification of the molecular mechanisms controlling regeneration-specific cellular events. A greater understanding of the process in regeneration-competent species may enable regeneration to be achieved in regeneration-incompetent species, including humans. Here we describe a protocol for labeling and repetitive imaging of aNSCs in vivo. We label single aNSCs, allowing nonambiguous re-identification of single cells in repetitive imaging sessions using electroporation of a red-reporter plasmid in Tg(gfap:GFP)mi2001 transgenic fish expressing GFP in aNSCs. We image using two-photon microscopy through the thinned skull of anesthetized and immobilized fish. Our protocol allows imaging every 2 d for a period of up to 1 month. This methodology allowed the visualization of aNSC behavior in vivo in their natural niche, in contrast to previously available technologies, which rely on the imaging of either dissociated cells or tissue slices. We used this protocol to follow the mode of aNSC division, fate changes and cell death in both the intact and injured zebrafish telencephalon. This experimental setup can be widely used, with minimal prior experience, to assess key factors for processes that modulate aNSC behavior. A typical experiment with data analysis takes up to 1.5 months.

Administration of Gemcitabine After Pancreatic Tumor Resection in Mice Induces an Antitumor Immune Response Mediated by Natural Killer Cells

Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D× p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon. We co-delivered a transposon encoding a constitutively active form of Akt2 (myrAkt2). Carcinogenesis and histopathologic features of tumors were examined. Metastasis was monitored by bioluminescence imaging. Tumors were resected and mice were given gemcitabine, and tumor recurrence patterns and survival were determined. Immune cells were collected from resection sites and analyzed by flow cytometry and in depletion experiments. After electroporation of oncogenic plasmids, mice developed a single pancreatic tumor nodule with histopathologic features of human PDAC. Pancreatic tumors that expressed myrAkt2 infiltrated the surrounding pancreatic tissue and neurons and became widely metastatic, reflecting the aggressive clinical features of PDAC in patients. Despite early tumor resection, mice died from locally recurring and distant tumors, but adjuvant administration of gemcitabine after tumor resection prolonged survival. In mice given adjuvant gemcitabine or vehicle, gemcitabine significantly inhibited local recurrence of tumors, but not metastasis to distant organs, similar to observations in clinical trials. Gemcitabine inhibited accumulation of CD11b+Gr1intF4/80int myeloid-derived suppressor cells at the resection margin and increased the number of natural killer (NK) cells at this location. NK cells but not Tcells were required for gemcitabine-mediated antitumor responses. Gemcitabine administration after resection of pancreatic tumors in mice activates NK cell-mediated antitumor responses and inhibits local recurrence of tumors, consistent with observations from patients with PDAC. Transgenic mice with resectable pancreatic tumors might be promising tools to study adjuvant therapy strategies for patients.

404. Development of Combinational Cellular Immunotherapy to Treat Pediatric Acute Myeloid Leukemia Patients

Hematopoietic stem cell transplantation is the standard of care for pediatric acute myeloid leukemia (AML) patients, but relapse still occurs in approximately 40% of AML patients. Hence there is an unmet need to improve disease control with fewer adverse acute and late effects. We therefore aim to develop ‘HLA-matched’ modified cellular therapies based on a platform of cord blood transplantation (CBT) to treat pediatric AML patients. In the majority of AML patients the tumor oncogene Wilms tumor 1 (WT1) is overexpressed and can serve as a target for tumor specific therapy. In addition to our previously developed cord blood-derived dendritic cell vaccine (CB-DC) we are currently developing gene modified T lymphocytes from the same CB-unit to specifically target WT1 expressing AML to support and increase the anti-leukemic effects of CBT. Lentiviral vectors were developed to express recombinant T-cell receptors (TCR) specifically recognizing HLA-A2+ restricted WT1 peptide. TCR expression was improved by codon-optimization of the cDNA sequence including cysteines in the alpha and beta chain constant domains. These WT1-specific TCR α/β-chains were incorporated in a single lentiviral vector linked by a ‘self-cleaving’ T2A peptide to improve efficiency. The order of the alpha and beta TCR chains, and inclusion of a furin cleavage site in the expression cassette did not enhance TCR expression in Jurkat and primary T cells derived from either peripheral blood or cord blood. Primary T cells were stimulated with T2 cells and CB-DCs pulsed with the specific peptide, which resulted in T cell activation as measured by CD69 and CD137 upregulation, as well as IFNgamma production. To reduce the risk of off-target antigen recognition of gene-modified T cells, and to further improve efficacy, CRISPR/Cas9 can be used to eliminate endogenous TCR expression. Cas9 protein and guide RNA (gRNA) were delivered to GFP positive reporter Jurkat cells using plasmid and mRNA electroporation (EP), integrating and non-integrating lentiviral vectors and adenoviral vectors with the highest efficiency of knockout being achieved by EP. Two gRNAs targeting the constant domains of the alpha chain and three targeting those of the beta chain resulted in similar knockout efficiencies in Jurkat cells (approximately 25%). Of note, combining the gRNAs did not result in higher knockout efficiencies. Gene-editing at the molecular level was confirmed by next generation sequencing. We envision that addition of TCR-engineered T cells (introduction of tumor-specific TCR and knockdown of endogenous TCR), derived from the same cord blood as used for the transplantation, will more efficiently and with improved safety prevent early relapse after CBT by targeting WT1 AML.

433. DNA Monoclonal Antibodies Target Influenza Virus In Vivo

Despite promising innovations, influenza vaccines and antiviral drugs fail to provide full protection from seasonal infection, and provide little defense against novel and potentially pandemic viral strains. Broadly cross-protective monoclonal antibodies have been developed with the aim of providing protection against highly divergent influenza viruses. However, the utility of delivering purified protein antibody as therapy or prophylaxis against influenza is limited, especially in pandemic settings. Use of gene therapy to generate monoclonal antibodies in vivo provides a simplified, flexible, and relatively inexpensive alternative to protein antibody treatment.In this study, we used intramuscular electroporation of plasmid DNA encoding immunoglobulin to express DNA monoclonal antibodies (DMAb) against influenza hemagglutinin (HA) surface protein in mice. Multiple aspects of plasmid construction, antibody design, and delivery were optimized to enhance expression of DMAb from muscle cells in vivo. We investigated multiple antibody clones, including the broadly-neutralizing anti-influenza-H1 antibody 5J8. The 5J8 DMAb was expressed at µg/mL levels in serum of both nude and immune-competent mice. Serum DMAb produced from muscle in vivo were functional in vitro - with the ability to bind influenza HA, block hemagglutination of red blood cells, and neutralize influenza virus. Serum DMAb expression levels approximate those required for protection. Influenza challenge studies of mice treated with 5J8 DMAb are underway.DMAb provide an important new approach to immune therapy. DNA has an excellent safety profile and averts challenges of pre-existing serology associated with many viral vectors. Here, we demonstrate that DNA can be used to deliver consistently high levels of potent monoclonal antibodies for protection against a viral pathogen.

CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock‐in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near‐maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation.

The avian embryo has a well-documented history as a model system for the study of neurogenesis, morphogenesis, and cell fate specification. This includes studies of the chicken inner ear that employ in ovo electroporation, in conjunction with the Tol2 system, to yield robust long-term transgene expression. Capitalizing on the success of this delivery method, we describe a modified version of the Tol2 expression vector that readily accepts the insertion of a microRNA-encoding artificial intron. This offers a strategy to investigate the possible roles of different candidate microRNAs in ear development by overexpression. Here, we describe the general design of this modified vector and the electroporation procedure. This approach is expected to facilitate phenotypic screening of candidate miRNAs to explore their bioactivity in vivo.

Massively parallel cis-regulatory analysis in the mammalian central nervous system.

Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell–derived organoids.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Cell type-specific manipulation with GFP-dependent Cre recombinase.

SummaryThere are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

Protection against dengue disease by synthetic nucleic acid antibody prophylaxis/immunotherapy.

Dengue virus (DENV) is the most important mosquito-borne viral infection in humans. In recent years, the number of cases and outbreaks has dramatically increased worldwide. While vaccines are being developed, none are currently available that provide balanced protection against all DENV serotypes. Advances in human antibody isolation have uncovered DENV neutralizing antibodies (nAbs) that are capable of preventing infection from multiple serotypes. Yet delivering monoclonal antibodies using conventional methods is impractical due to high costs. Engineering novel methods of delivering monoclonal antibodies could tip the scale in the fight against DENV. Here we demonstrate that simple intramuscular delivery by electroporation of synthetic DNA plasmids engineered to express modified human nAbs against multiple DENV serotypes confers protection against DENV disease and prevents antibody-dependent enhancement (ADE) of disease in mice. This synthetic nucleic acid antibody prophylaxis/immunotherapy approach may have important applications in the fight against infectious disease.

Association of astrocytes with neurons and astrocytes derived from distinct progenitor domains in the subpallium.

Astrocytes play pivotal roles in metabolism and homeostasis as well as in neural development and function in a manner thought to depend on their region-specific diversity. In the mouse spinal cord, astrocytes and neurons, which are derived from a common progenitor domain (PD) and controlled by common PD-specific transcription factors, migrate radially and share their final positions. However, whether astrocytes can only interact with neurons from common PDs in the brain remains unknown. Here, we focused on subpallium-derived cells, because the subpallium generates neurons that show a diverse mode of migration. We tracked their fate by in utero electroporation of plasmids that allow for chromosomal integration of transgenes or of a Cre recombinase expression vector to reporter mice. We also used an Nkx2.1Cre mouse line to fate map the cells originating from the medial ganglionic eminence and preoptic area. We find that although neurons and astrocytes are labeled in various regions, only neurons are labeled in the neocortex, hippocampus and olfactory bulb. Furthermore, we find astrocytes derived from an Nkx 2.1-negative PD are associated with neurons from the Nkx2.1+ PD. Thus, forebrain astrocytes can associate with neurons as well as astrocytes derived from a distinct PD.

Use of heterodimeric IL-15 in immune therapeutic regimens against HIV-1 or SIV infection (VAC8P.1048)

Abstract Interleukin-15 (IL-15) promotes the growth of NK and memory CD8 T cells, which makes it an attractive candidate in immune therapeutic strategies to eradicate HIV-1 infection. We have shown that the relevant IL-15 molecule in vivo is the heterodimer (hetIL-15) composed of IL-15 chain and the so-named IL-15 Receptor alpha (IL-15Ra) chain. We developed expression vectors producing high levels of bioactive hetIL-15. We have also developed stable cell lines overproducing IL-15 heterodimers. SIV-infected and uninfected rhesus macaques were treated with these DNA plasmids by electroporation. In addition, SHIV-infected macaques received repeated subcutaneous injections of purified hetIL-15 protein or placebo. Electroporation of plasmid DNA resulted in increased levels of circulating IL-15, which typically peaked between day 4 and 5 in all the animals. Similarly, treatment with hetIL-15 protein gave sustained high cytokine plasma levels without any significant changes in plasma VL or any other adverse effects. In both studies, increased levels of IL-15 resulted in early decrease in the percentage of NK cells, probably linked to their mobilization out of the bloodstream, and preferential accumulation of CD8+ T lymphocytes with effector memory phenotype. In conclusion, the combination of CD8 effector cells expansion and lack of viral activation makes IL-15 an ideal component in immune therapeutic strategies for the treatment and potential eradication of HIV-1 infection.

Strategies for Skeletal Muscle Targeting in Drug Discovery

The targeting of drugs to skeletal muscle is an emerging area of research. Driven by the need for new therapies to treat a range of muscle-associated diseases, these strategies aim to provide improved drug exposure at the site of action in skeletal muscle with reduced concentration in other tissues where unwanted side effects could occur. By interacting with muscle-specific cell surface recognition elements, both tissue localization and selective uptake into skeletal muscle cells can be achieved. The design of molecules that are substrates for muscle uptake transporters can provide concentration in m uscle tissue. For example, drug conjugates with carnitine can provide improved muscle uptake via OCTN2 transport. Binding to muscle surface recognition elements followed by endocytosis can allow even large molecules such as antibodies to enter muscle cells. Monoclonal antibody 3E10 demonstrated selective uptake into skeletal muscle in vivo. Hybrid adeno-associated viral vectors have recently shown promise for high skeletal muscle selectivity in gene transfer applications. Delivery technology methods, including electroporation of DNA plasmids, have also been investigated for selective muscle uptake. This review discusses challenges and opportunities for skeletal muscle targeting, highlighting specific examples and areas in need of additional research.

Curing genetic mutations for the next generation: CRISPR/Cas9-mediated gene correction in mouse spermatogonial stem cells

The CRISPR/Cas system is a very powerful and versatile tool for genomic manipulation. A recent study published in Cell Research by Wu et al. [1] reported a Cas9-mediated strategy to correct a disease-causing mutation in the mouse gammaC-crystallin(Crygc) locus in spermatogonial stem cells (SSCs) to cure the resulting cataracts disease in the offspring with 100 % efficiency. Importantly, no off-target mutations were identified through whole-genome sequencing. This study is a perfect combination of three different high biological technologies, SSCs manipulation, CRISPR/ Cas gene editing and round spermatid injection (ROSI), shedding light on clinical applications for repair of genetic mutations in the progeny. Many genetic disorders are caused by DNA mutation(s) which is usually inherited from a parental genome and potentially passes to the next generation. Although some symptoms of certain genetic diseases are alleviated with medical or surgical treatments, the disease is hard to cure due to limitations of available approaches to manipulate the human genome efficiently. In recent years, with the emergence of programmable nucleases, scientists are able to edit specific genomic sites by creating DNA doublestrand breaks (DSBs) which stimulate DNA repair either through nonhomologous end joining (NHEJ) or homologydirected repair (HDR) pathways depending on the presence of the repair template [2]. Compared with zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system is more popular due to its simplicity, efficiency and flexibility [3]. The CRISPR/Cas system is derived from the adaptive immune system in bacteria and archaea [4]. It consists of the endonuclease Cas9 (from S. pyogenes) and a customized single-guide RNA (sgRNA) that leads the Cas9 to the DNA target following base-paring rules [4]. With the CRISPR/ Cas system, researchers have demonstrated quick generation of gene-edited model organisms [5, 6] or correction of genetic mutations in animals [7, 8]. Although both somatic and one-cell embryo injection of CRISPR/Cas-mediated gene repair alleviates disease symptoms, these studies neither could cure the disease nor avoid off-target digestion of Cas9 endonuclease. More importantly, the mutations were still transmissable to the next generation. Spermatogonial stem cells (SSCs) are self-renewing male germ line stem cells that are responsible for producing numerous spermatozoa and transmitting genetic information through fertilization. The techniques of in vitro culture and proliferation of SSCs from different species including humans have been well developed [9, 10]. Recently, by taking advantages of SSC manipulation, the CRISPR/Cas system and ROSI, Wu et al. took the lead in gene editing in mouse SSCs and the generation of genetically modified animal models. Moreover, they also used the strategy to correct a genetic disease in the new born pups with 100 % efficiency [1]. To test the feasibility, Wu et al. first disrupted an eGFP transgene in the SSCs in vitro by electroporation of plasmids expressing Cas9 and sgRNA targeting eGFP. The SSCs derived from actin-eGFP transgenic male mice were stably transduced with an mRFP marker to facilitate tracing. Two weeks after transfection of Cas9–sgRNA plasmids, the SSCs expressing mRFP alone were sorted. The sorted SSCs were transplanted into the seminiferous tubules of busulfan-treated males. Round spermatids from the recipient males were isolated and injected into wildtype oocytes to produce mice. After sequencing, half of the D. Li (&) Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China e-mail: dlli@bio.ecnu.edu.cn

CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells for adoptive therapy

Meeting abstracts Strategies that enhance the function of T cells are critical for immunotherapy. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids

High-efficiency electroporation of the spinal cord in larval axolotl.

Axolotls are well known for their remarkable ability to regenerate complex body parts and structures throughout life, including the entire limb and tail. Particularly fascinating is their ability to regenerate a fully functional spinal cord after losing the tail. Electroporation of DNA plasmids or morpholinos is a valuable tool to gain mechanistic insight into the cellular and molecular basis of regeneration. It provides among other advantages a simple and fast method to test gene function in a temporally and spatially controlled manner. Some classic drawbacks of the method, such as low transfection efficiency and damage to the tissue, had hindered our understanding of the contribution of different signaling pathways to regeneration. Here, we describe a comprehensive protocol for electroporation of the axolotl spinal cord that overcomes this limitations using a combination of high-voltage and short-length pulses followed by lower-voltage and longer-length pulses. Our approach yields highly efficient transfection of spinal cord cells with minimal tissue damage, which now allows the molecular dissection of spinal cord regeneration.