Cell-specific Promoter Research Articles

Genetic manipulation of the genes for clonal seeds results in sterility in cotton

BackgroundHeterosis is a common phenomenon in plants and has been extensively applied in crop breeding. However, the superior traits in the hybrids can only be maintained in the first generation but segregate in the following generations. Maintaining heterosis in generations has been challenging but highly desirable in crop breeding. Recent study showed that maternally produced diploid seeds could be achieved in rice by knocking out three meiosis related genes, namely REC8, PAIR1, OSD1 to create MiMe in combination with egg cell specific expression of BBM transcription factor, a technology called clonal seeds. Interestingly, there has been very limited reports indicating the feasibility of this approach in other crops.ResultsIn this study, we aimed to test whether clonal seeds could be created in cotton. We identified the homologs of the three meiosis related genes in cotton and used the multiplex CRISPR/Cas9 gene editing system to simultaneously knock out these three genes in both A and D sub-genomes. More than 50 transgenic cotton plants were generated, and fragment analysis indicated that multiple gene knockouts occurred in the transgenic plants. However, all the transgenic plants were sterile apparently due to the lack of pollen. Pollination of the flowers of the transgenic plants using the wild type pollens could not generate seeds, an indication of defects in the formation of female sexual cells in the transgenic plants. In addition, we generated transgenic cotton plants expressing the cotton BBM gene driven by the Arabidopsis egg cell specific promoter pDD45. Two transgenic plants were obtained, and both showed severely reduced fertility.ConclusionsOverall, our results indicate that knockout of the clonal seeds related genes in cotton causes sterility and how to manipulate genes to create clonal seeds in cotton requires further research.

Partial chemogenetic inhibition of the locus coeruleus due to heterogeneous transduction of noradrenergic neurons preserved auditory salience processing in wild-type rats.

The acoustic startle reflex (ASR) and prepulse inhibition of the ASR (PPI) assess the efficiency of salience processing, a fundamental brain function that is impaired in many psychiatric conditions. Both ASR and PPI depend on noradrenergic transmission, yet the modulatory role of the locus coeruleus (LC) remains controversial. Clonidine (0.05 mg/kg, i.p.), an alpha2-adrenoreceptor agonist, strongly reduced the ASR amplitude. In contrast, chemogenetic LC inhibition only mildly suppressed the ASR and did affect the PPI in virus-transduced rats. The canine adenovirus type 2 (CAV2)-based vector carrying a gene cassette for the expression of inhibitory receptors (hM4Di) and noradrenergic cell-specific promoter (PRSx8) had high cell-type specificity (94.4±3.1%) but resulted in heterogeneous virus transduction of DbH-positive LC neurons (range: 9.2-94.4%). Clozapine-N-oxide (CNO; 1mg/kg, i.p.), a hM4Di actuator, caused the firing cessation of hM4Di-expressing LC neurons, yet complete inhibition of the entire population of LC neurons was not achieved. Case-based immunohistochemistry revealed that virus injections distal (> 150μm) to the LC core resulted in partial LC transduction, while proximal (< 50 μm) injections caused neuronal loss due to virus neurotoxicity. Neither the ASR nor PPI differed between the intact and virus-transduced rats. Our results suggest that a residual activity of virus-non-transduced LC neurons might have been sufficient for mediating an unaltered ASR and PPI. Our study highlights the importance of a case-based assessment of the virus efficiency, specificity, and neurotoxicity for targeted cell populations and of considering these factors when interpreting behavioral effects in experiments employing chemogenetic modulation.

Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

Establishment of a humanized ST6GAL1 mouse model for influenza research.

This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1. Humanized fragments, consisting of the endothelial cell-specific K18 promoter, human ST6GAL1-encoding gene, and luciferase gene, were microinjected into the fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offspring were identified using PCR. Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation, and invivo analysis was performed for screening. Expression of the humanized gene was tested by performing immunohistochemical (IHC) analysis. Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed. Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1. Seven mice were identified as carrying copies of the humanized gene, and the invivo analysis indicated that hST6GAL1 gene expression in positive mice mirrored influenza virus infection characteristics. The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice. Moreover, the hematologic and biochemical parameters of the positive mice were within the normal range. A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

From Genome-wide Association Studies to Functional Variants: ARL14 Cis-regulatory Variants Are Associated With Severe Malaria.

Genome-wide association studies have identified several nonfunctional tag single-nucleotide polymorphisms (SNPs) associated with severe malaria. We hypothesized that causal SNPs could play a significant role in severe malaria by altering promoter or enhancer activity. Here, we sought to identify such regulatory SNPs. SNPs in linkage disequilibrium with tagSNPs associated with severe malaria were identified and were further annotated using FUMA. Then, SNPs were prioritized using the integrative weighted scoring method to identify regulatory ones. Gene reporter assays were performed to assess the regulatory effect of a region containing candidates. The association between SNPs and severe malaria was assessed using logistic regression models in a Senegalese cohort. Among 418 SNPs, the best candidates were rs116525449 and rs79644959, which were in full disequilibrium between them, and located within the ARL14 promoter. Our gene reporter assay results revealed that the region containing the SNPs exhibited cell-specific promoter or enhancer activity, while the SNPs influenced promoter activity. We detected an association between severe malaria and those 2 SNPs using the overdominance model and we replicated the association of severe malaria with the tagSNP rs116423146. We suggest that these SNPs regulate ARL14 expression in immune cells and the presentation of antigens to T lymphocytes, thus influencing severe malaria development.

Abstract 7254: Evaluation of scFv and diabody pseudotyped Sindbis engineered retrovectors for targeted cancer therapy

Abstract Engineering viral vectors to target specific cells and organs after intravenous delivery remains a challenge. Transcriptional targeting with cell-specific promoters is a commonly used approach but does not change vector biodistribution. We have developed a novel gene delivery system targeted to tumor cells by pseudotyping the vector with Sindbis Virus (SB) envelope bearing an antigen-binding domain. Prototype vectors were generated by inserting a universal IgG binding domain from Protein A into the SB envelope (SB-ZZ). Antibodies (Abs) against various cell-surface targets (HLA, HER2, CD47, EGFR, Nectin-4), were conjugated to SB-ZZ vectors delivering a luciferase (Luc) reporter gene. Targeted transduction was confirmed on cells expressing the corresponding antigens by significantly higher Luc activity compared to SB-ZZ vectors without Abs. SB envelopes were further engineered by adding or removing furin cleavage sites and linkers, transduction-enhancing point mutations, and direct incorporation of single-chain variable fragment (scFv) in different VH and VL orientations, or diabody (DB) sequences, against the same targets. The specificity of optimized HER2-targeting vectors delivering the Luc reporter was evaluated in SKBR3 (HER2 over-expressing) and MDA-MB-468 (HER2-null) cells. Robust Luc activity was observed in SKBR3 cells, but only negligible activity in MDA-MB-468 cells. In MDA-MB-468 cells overexpressing HER2, significantly higher transduction was observed compared to the wild-type cells. When comparing HER2-targeting vectors with scFv vs. DB sequences based on the same antibody, transduction capability was higher with the DB motif in HER2-positive cell lines. Targeting vectors delivering HSV-eTK were also constructed, and ganciclovir (GCV)-mediated cell killing was evaluated in vitro. SKBR3 cells transduced with HER2-targeting vectors showed significantly more cell killing after GCV treatment than after transduction with an amphotropic reference vector. Our results indicate a method for directing gene therapy vectors to specific populations of tumor cells, and provide a framework for highly modular targeted gene therapy and immunotherapeutics. Experiments are underway to examine in vivo delivery of targeted vs amphotropic vectors. Citation Format: Makoto Sato, Alexander Cunha, Sara Collins, Noriyuki Kasahara, Cecilia Roh, Robert G. Johnson. Evaluation of scFv and diabody pseudotyped Sindbis engineered retrovectors for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7254.

Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.

The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.

Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear.

Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a "multiple vectors in one AAV" strategy for promoter construction invivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof -/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.

Injectable Supramolecular Hydrogels for In Situ Programming of Car-T Cells toward Solid Tumor Immunotherapy.

Chimeric antigen receptor (CAR)-T cell immunotherapy is approved in the treatment of hematological malignancies, but remains far from satisfactoryin solid tumor treatment due to inadequate intra-tumor CAR-T cell infiltration. Herein, an injectable supramolecular hydrogel system, based on self-assembly between cationic polymer mPEG-PCL-PEI (PPP) conjugated with T cell targeting anti-CD3e f(ab')2 fragment and α-cyclodextrin (α-CD), is designed to load plasmid CAR (pCAR) with a T cell specific CD2 promoter, which successfully achieves in situ fabrication and effective accumulation of CAR-T cells at the tumor site in humanized mice models. More importantly, due to this tumor microenvironment reprogramming, secretion of cellular inflammatory cytokines (interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) or tumor killer protein granzyme B is significantly promoted, which reverses the immunosuppressive microenvironment and significantly enhances the intra-tumor CAR-T cells and cytotoxic T cells infiltration. To the best of the current knowledge, this is a pioneer report of using injectable supramolecular hydrogel for in situ reprogramming CAR-T cells, which might be beneficial for solid tumor CAR-T immunotherapy.

A Mouse Model of CD19 CAR - T Cell Therapy Reveals Mechanism of Cytopenia

Immunotherapy with Chimeric Antigen Receptor (CAR) T cells against CD19 can cure patients with refractory B cell lymphoma and leukemia. However, complications of CAR-T cell therapy can cause morbidity. Among the complications of CAR-T cell therapy is cytopenia, i.e. decreased peripheral blood counts of platelets, leukocytes and / or erythrocytes. The exact mechanism of this side - effect is not known, retrospective analysis showed it affects 10-20% of patients and is linked with the severity of cytokine release syndrome (Juluri et al, PMID34666344). We have previously developed a mouse model of CAR-T cell therapy using C56BL/6 mouse expressing human CD19 under a B- Cell specific promoter (Pennell et al, PMID29735365). The recipient mice are heterozygous for human hCD19 on CD45.2 background. Congenic C56BL/6 CD45.1 mice served as T cell donors. The aim of this study was to investigate if the mouse model recapitulates cytopenia seen in patients after CAR-T cell therapy. C56BL/6 recipient mice were treated with lymphodepleting chemotherapy (Cyclophosphamide (CY) 300 mg/kg) and infused with 1) no T cells 2) control T cells transfected with hEGFR 3) T cells transfected with CD19 CAR with hCD137 and hCD3zeta signaling domains. The chosen dose of infused T cells at 0.3E+6 per animal was low, approximately 10x below the lethal dose and 3x less than IC50 dose, to reduce early mortality. The animals were weighed and examined regularly, and appearance and activity were scored from 0 (healthy) to 8 (moribund). Total of 30 animals per group were harvested at 5 time points, 6 mice each, on days 5, 12, 26, 40 and 54. Peripheral blood counts were measured using HemaVet CBC counter. Flow cytometry was performed on blood and bone marrow. Spleens, long bones (femur, tibia) and sternum were harvested for histopathologic assessment. Multiparametric immunohistochemistry (IHC) was performed using Codex (Phenocycler, Akoya). The mice receiving CAR-19 T cells showed significant, but transient loss of weight and transient increase in morbidity score during the first 2 weeks. Mortality was not significantly different between groups: 0, 1 and 2 deceased animals in CY, control T and CAR-T cell groups, respectively. We observed transient significant cytopenia in the CAR-T cell treated mice, with significantly lower platelet counts on day 5 (p&amp;lt;0.05) and lower White Blood Cell counts on days 12 (p&amp;lt;0.06) and day 26 (p&amp;lt;0.05). These changes were associated with 100-fold larger expansion of CAR-T cells in blood as compared to the control hEGFR T cells and 20-fold higher relative expansion of CAR-T cells in the bone marrow. Multiparametric IHC showed loss of B cell follicles and increased density of donor T cells in the CAR-T cells recipients compared to hEGFR T cell control. Pathologic evaluation of sternums revealed individual segments (sternebra) with necrosis of the hematopoietic elements at the first time point (day 5). At subsequent time points there were progressive changes reminiscent of evolution of infarct with organization, proliferation of fibroblasts and fibrosis with increased osteogenesis and finally with regression of mesenchymal elements and reestablishment of hematopoiesis (Figure 1). The number of segments with necrosis and sequalae was significantly higher in the CAR-T cell group as compared to the hEGFR T cell control (p=0.0005). The necrosis of hematopoietic elements within individual sternum segments involved at the most 3 segments out of 6. The involved segments appeared to distribute randomly: either adjacent or separated by viable segments. The necrotic segments showed paucity of erythrocytes with foci of recent erythrocyte extravasation consistent with ischemic infarct. The mouse model of CAR-T cell therapy against CD19 recapitulates cytopenia in early post - treatment period. The observed pattern of necrosis of the hematopoietic elements in entire segments suggests compromised blood supply. We hypothesize sternebra are more susceptible to necrosis due to limited blood supply, separate for each segment. We hypothesize that occlusion of arteries or capillaries in the bone marrow may contribute to the mechanism of cytopenia following CAR-T cell therapy. Prior retrospective analysis linked the cytopenia with increased levels of inflammatory mediators of Cytokine Release Syndrome. The present findings highlight a role of local factors in situ, which may be influenced by systemic cytokines.

Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate.

OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

Isolation and characterisation of promoters from mouse genome to drive post-meiotic germ cell-specific robust gene expression for functional genomics studies

The generation of spermatozoa from developing germ cells through mitotic and meiotic divisions is a highly regulated and complex process. Any defect in this process, may lead to subfertility/infertility. The role of different transcripts (mRNA/miRNA/lncRNA) in regulation of the pre-meiotic, meiotic, and post-meiotic stages of spermatogenesis are being proposed based on various multiomics based approaches. Such differential gene-expression is regulated by promoter elements that are activated in a stage specific manner. To determine the role of these differentially expressed transcripts in the process of meiosis, a robust post-meiotic germ cell specific promoter is required. In the present study, we have isolated and characterized the expression of the mouse Proacrosin, SP10, and ELP promoters for driving post-meiotic germ cell specific gene-expression. Promoter regions of all these 3 genes were isolated and cloned to generate mammalian expression vector. The transgene expression in post-meiotic germ cells was assessed in mice using the testicular electroporation method in vitro as well as in vivo, using above promoters. It was also validated in goat seminiferous tubules, in vitro. We have also carried out a comparative analysis of the strength of these promoters to confirm their robustness that indicated Proacrosin to be the most robust promoter that can be useful for diving post-meiotic germ cells specific gene-expression. These promoters can be used to alter gene-expression specifically in post-meiotic germ cells for deciphering the role(s) of germ cell genes in spermatogenic progression or for expressing various genome editing tools for engineering the germ cell genome to understand basis of subfertility/infertility.

Preclinical evaluation of the efficacy and safety of AAV1-hOTOF in mice and nonhuman primates

Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual AAV1 carrying human OTOF coding sequences with the expression driven by the hair cell-specific promoter Myo15, AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models by pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof–/– mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea although it was detected in other organs such as the central nervous system and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60–94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.

Adenoviral vectors infect B lymphocytes in vivo

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized exvivo modification of the cells. Direct invivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of exvivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both exvivo and invivo. We found that unmodified human adenovirus C5 was capable of infecting B cells invivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection invivo.

CRISPR/Cas9 Based Cell-Type Specific Gene Knock-Out in Arabidopsis Roots.

CRISPR/Cas9 (hereafter Cas9)-mediated gene knockout is one of the most important tools for studying gene function. However, many genes in plants play distinct roles in different cell types. Engineering the currently used Cas9 system to achieve cell-type-specific knockout of functional genes is useful for addressing the cell-specific functions of genes. Here we employed the cell-specific promoters of the WUSCHEL RELATED HOMEOBOX 5 (WOX5), CYCLIND6;1 (CYCD6;1), and ENDODERMIS7 (EN7) genes to drive the Cas9 element, allowing tissue-specific targeting of the genes of interest. We designed the reporters to verify the tissue-specific gene knockout in vivo. Our observation of the developmental phenotypes provides strong evidence for the involvement of SCARECROW (SCR) and GIBBERELLIC ACID INSENSITIVE (GAI) in the development of quiescent center (QC) and endodermal cells. This system overcomes the limitations of traditional plant mutagenesis techniques, which often result in embryonic lethality or pleiotropic phenotypes. By allowing cell-type-specific manipulation, this system has great potential to help us better understand the spatiotemporal functions of genes during plant development.

Design and deep learning of synthetic B- cell-specific promoters

Design and deep learning of synthetic B- cell-specific promoters

Cardiac PDGFRα+ interstitial cells generate spontaneous inward currents that contribute to excitability in the heart.

The cell types and conductance that contribute to normal cardiac functions remain under investigation. We used mice that express an enhanced green fluorescent protein (eGFP)-histone 2B fusion protein driven off the cell-specific endogenous promoter for Pdgfra to investigate the distribution and functional role of PDGFRα+ cells in the heart. Cardiac PDGFRα+ cells were widely distributed within the endomysium of atria, ventricle, and sino-atrial node (SAN) tissues. PDGFRα+ cells formed a discrete network of cells, lying in close apposition to neighboring cardiac myocytes in mouse and Cynomolgus monkey (Macaca fascicularis) hearts. Expression of eGFP in nuclei allowed unequivocal identification of these cells following enzymatic dispersion of muscle tissues. FACS purification of PDGFRα+ cells from the SAN and analysis of gene transcripts by qPCR revealed that they were a distinct population of cells that expressed gap junction transcripts, Gja1 and Gjc1. Cardiac PDGFRα+ cells generated spontaneous transient inward currents (STICs) and spontaneous transient depolarizations (STDs) that reversed at 0 mV. Reversal potential was maintained when ECl = -40 mV. [Na+ ]o replacement and FTY720 abolished STICs, suggesting they were due to a non-selective cation conductance (NSCC) carried by TRPM7. PDGFRα+ cells also express β2 -adrenoceptor gene transcripts, Adrb2. Zinterol, a selective β2 -receptor agonist, increased the amplitude and frequency of STICs, suggesting these cells could contribute to adrenergic regulation of cardiac excitability. PDGFRα+ cells in cardiac muscles generate inward currents via an NSCC. STICs generated by these cells may contribute to the integrated membrane potentials of cardiac muscles, possibly affecting the frequency of pacemaker activity.

Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange and drug selection.

I outline a streamlined method to insert large, single-copy transgenes into the Caenorhabditis elegans genome using recombination-mediated cassette exchange (RMCE) that relies solely on drug selection yielding a homozygous fluorescent protein (FP) marked transgene in 3 generations (8 days) at high efficiency (>1 insertion per 2 injected P0 animals). Landing sites for this approach are available on four chromosomes in several configurations which yield lines marked in distinct cell types. An array of vectors permit creating transgenes using a variety of selection methods (HygR, NeoR, PuroR, and unc-119) that yield lines expressing different colored FPs (BFP, GFP, mNG, and Scarlet). Although these transgenes retain a plasmid backbone and a selection marker, the inclusion of these sequences typically does not alter the expression of several cell-specific promoters tested. However, in certain orientations, promoters exhibit crosstalk with adjacent transcription units. In cases where crosstalk is problematic, the loxP-flanked fluorescent marker, plasmid backbone, and hygR gene can be excised by crossing through germline Cre expressing lines also created using this technique. Finally, genetic and molecular reagents designed to facilitate customization of both targeting vectors and landing sites are also described. Together, the rapid RMCE toolbox provides a platform for developing further innovative uses of RMCE to create complex genetically engineered tools.

Expression of a truncated maize SPS increases source capacity in maize

AbstractIn an attempt to increase source capacity, transgenic corn was generated by expressing a truncated maize sucrose phosphate synthase (ZmSPSΔ482) under two leaf mesophyll cellspecific promoters (CAB and PPDK). The endogenous and truncated SPS proteins from transgenic leaf extracts were distinguishable by protein immunoblot analysis. The expression of transgenic SPS protein across events varied from very low to very high and included several cosuppressed events. SPS activity showed a diurnal pattern in both transgenic and wild-type maize leaves. In greenhouse experiments, transgenic maize had higher leaf sucrose and lower leaf starch, suggesting a shift in carbon partitioning from starch to sucrose. Conversely, cosuppressed events had lower leaf sucrose and higher leaf starch. A field test was performed to compare sucrose and starch in positive and negative isolines of hybrid maize CAB and PPDK ZmSPSΔ482 events. In the field, many positive isolines had higher levels of both leaf sucrose and starch than the negative isolines. This suggests that in the field, with higher light intensity the shift in carbon partitioning from starch to sucrose, observed under greenhouse conditions did not occur. This in turn suggests that the environment affects the phenotype of the transgenics and that in the field, there was an overall increase in carbon assimilation. Six events from each construct were tested in a pilot multi-density yield trial but overall, no effect on yield was observed. Therefore, although the transgenic plants had more source capacity, this did not translate into higher seed yield.

CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision

Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology—in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)—to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.