Manipulation Of Cells Research Articles

Highly Parallel and High‐Throughput Nanoliter‐Scale Liquid, Cell, and Spheroid Manipulation on Droplet Microarray

AbstractThe droplet microarray (DMA) platform is a powerful tool for high‐throughput biological and chemical applications, enabling miniaturization and parallelization of experimental processes. Capable of holding hundreds of nanoliter droplets, it facilitates the screening and analysis of samples, such as cells, bacteria, embryos, and spheroids. Handling thousands of small volumes in parallel presents significant challenges. In this study, we utilize the open format of the DMA for controlled, parallel high‐throughput liquid manipulations using the sandwich technique. We demonstrate high‐throughput medium replacement at nanoliter‐scale, maintaining high cell viability on DMA for up to 7 days; for HeLa‐CLL2 cells (adherent) and SU‐DHL4 cells (suspension), and up to 14 days for HEK293 spheroids. Additionally, we achieve highly parallel aliquot uptake from nanoliter droplets, enabling non‐destructive cell viability assessments. Furthermore, the presented method enables the parallel transfer of cell spheroids between different DMAs, allowing transfer and pooling of spheroids in seconds without damage. These advances significantly enhance the capabilities of the DMA platform, enabling long‐term cell culture in nanoliter droplets and parallel sampling for high‐throughput cell or spheroid manipulation. This broadens the scope of DMA's potential applications in fields such as cell‐based high‐throughput screening, formation of complex 3D cell models for drug screening, and microtissue engineering.

Classification of Cell Therapy Products by Cell Manipulation Degree and Functions Performed: Analysis of International Regulatory Approaches

INTRODUCTION. The degree of processing (manipulation) of cells included in a cell product and the functions performed after administration (homologous/non-homologous use) determine the classification of the cell product as a transplant or an advanced therapy medicinal product (ATMP) and, hence, the regulatory aspects of the product’s life cycle. Currently, the legislation of the Eurasian Economic Union (EAEU) and the Russian Federation does not sufficiently explain the terms ‘minimal manipulation’ and ‘homologous/non-homologous use’, which may lead to the use of cell products with unproven safety and efficacy in humans.AIM. This study aimed to compare Russian and international approaches to the interpretation of the terms ‘minimal manipulation’ and ‘homologous/non-homologous use’ for classifying cell products and determining their regulatory pathways, with stromal vascular fraction (SVF) products used as an example.DISCUSSION. This article reviews and summarises the regulatory approaches of the Russian Federation, the EAEU, the United States (US), and the European Union (EU) that are based on the classification of cell products according to the degree of cell manipulation and the functions performed after administration. The authors have analysed and compared the regulatory acts and approaches of the countries under consideration, with SVF products as a case study. The article highlights general aspects of interpreting the terms ‘minimal manipulation’ and ‘homologous/ non-homologous use’ and demonstrates the difference in regulatory approaches across several countries, which lies in the classification of enzymatic processing and selective collection of cells as substantial or minimal manipulation.CONCLUSIONS. The mechanism for regulating cell products depends on the degree of cell manipulation (substantial or minimal) and the intended use (homologous or non-homologous). A common principle adopted by regulatory agencies in the US, EU, EAEU, and Russia is to classify manipulation as minimal if the manipulated cells preserve their biological characteristics and physiological function. A defining characteristic of the homologous use of cells or tissues is their administration to perform their inherent functions in the body. In Russia, the regulatory acts for ATMPs and for transplants list the procedures classified as minimal manipulation. According to international standards, preparations based on minimally manipulated SVF cells are classified as ATMPs when used non-homologously. The lack of comprehensive and clear explanations of the terms ‘minimal manipulation’ and ‘homologous/non-homologous use’ in the legislation of the EAEU and the Russian Federation necessitates the development of relevant guidelines providing specific examples.

Cysteamine functionalized gold nanoparticles exhibit high efficiency delivery of genetic materials in embryonic stem cells majorly via clathrin mediated endocytosis

Efficient and safe gene delivery is vital for genetic manipulation of stem cells for regenerative medicine. Gold nanoparticles have been used for various biomedical applications in the past, and are currently being researched as transfection agents. In this study, we report a simple one-pot synthesis of positively charged gold nanoparticles functionalized with cysteamine. The nanoparticles exhibit no cytotoxicity and can bind to both plasmid DNA (pDNA) as well as small interference RNA (siRNA). We observed that a five fold lower concentration of pDNA was sufficient for achieving comparable overexpression as that of a commercial transfection agent. We also observed that about 70 % transient silencing of the target gene was achieved with only 25 nM siRNA delivered by our nano-vehicle. To better understand the fate of the nanoparticle, we attempted to identify its uptake mechanism. The results indicate that while all the mechanisms contribute to the uptake, the clathrin-dependent pathway plays a major role. This is the first study on understanding the mechanism of uptake of CA-AuNPs conjugated to pDNA by embryonic stem cells. This is also the first study, where a successful transfection using gold based nanoparticles has been achieved in ESCs at a concentration as low as 0.5 µg/ml for pDNA and 25ƞM siRNA.

The Phytophthora infestans effector Pi05910 suppresses and destabilizes host glycolate oxidase StGOX4 to promote plant susceptibility.

Phytophthora infestans is a notorious oomycete pathogen that causes potato late blight. It secretes numerous effector proteins to manipulate host immunity. Understanding mechanisms underlying their host cell manipulation is crucial for developing disease resistance strategies. Here, we report that the conserved RXLR effector Pi05910 of P. infestans is a genotype-specific avirulence elicitor on potato variety Longshu 12 and contributes virulence by suppressing and destabilizing host glycolate oxidase StGOX4. By performing co-immunoprecipitation, yeast-two-hybrid assays, luciferase complementation imaging, bimolecular fluorescence complementation and isothermal titration calorimetry assays, we identified and confirmed potato StGOX4 as a target of Pi05910. Further analysis revealed that StGOX4 and its homologue NbGOX4 are positive immune regulators against P. infestans, as indicated by infection assays on potato and Nicotiana benthamiana overexpressing StGOX4 and TRV-NbGOX4 plants. StGOX4-mediated disease resistance involves enhanced reactive oxygen species accumulation and activated the salicylic acid signalling pathway. Pi05910 binding inhibited enzymatic activity and destabilized StGOX4. Furthermore, mutagenesis analyses indicated that the 25th residue (tyrosine, Y25) of StGOX4 mediates Pi05910 binding and is required for its immune function. Our results revealed that the core RXLR effector of P. infestans Pi05910 suppresses plant immunity by targeting StGOX4, which results in decreased enzymatic activity and protein accumulation, leading to enhanced plant susceptibility.

ADVANCEMENTS IN REGENERATIVE MEDICINE: PRESENT APPROACHES, EMERGING STRATEGIES, AND FUTURE PERSPECTIVES

Background: Regenerative medicine is an evolving field with tremendous promise for treating various diseases and medical conditions. This innovative discipline encompasses various techniques, including the utilization of stem cells, tissue engineering, and gene therapy, to effectively regenerate or replace damaged tissues and organs. The rapid advancements in regenerative medicine over recent years have created new hope for patients who face otherwise untreatable conditions, showcasing the fields potential to transform healthcare. Objectives: The primary objective of this article is to provide a comprehensive overview of the current state of regenerative medicine. By delving into recent advancements and emerging technologies, this article aims to highlight the various FDA-approved therapies that have been developed to treat diverse pathologies. Additionally, it seeks to identify future directions for research and application within this dynamic field, ensuring that readers are informed about the latest breakthroughs and potential developments. Findings: To date, regenerative medicine has resulted in numerous FDA-approved therapies that target various pathologies. Extensive research has led to the creation of sophisticated grafts that leverage advanced scaffolding materials and cutting-edge cell manipulation technologies. These innovations allow for precise control over cell behavior and enhance tissue repair mechanisms. Strategies such as the controlled release of growth factors and vascular cell seeding are currently being developed to improve graft integration with both the host vasculature and the nervous system. Furthermore, there are ongoing efforts to stimulate and enhance the bodys healing response through immune system modulation, alongside the development of new cell sources for transplantation to address the persistent challenge of limited cell supply. Significance: The significance of regenerative medicine lies in its transformative potential to revolutionize the treatment of conditions that have long been deemed incurable. By providing insights into the advancements in graft technology, cell sourcing, and integration strategies, this article underscores the ultimate promise of regenerative medicine as a vital area of research. The findings and discussions presented here not only illuminate the current landscape but also pave the way for future innovations that could lead to groundbreakingtherapies, enhancing the quality of life for countless patients worldwide.[1][2].This article provides an overview and insights into the current state of the development of regenerative medicine and its various potential guiding into future directions.

Effects of Clump Size on the Pluripotency and Proliferation in the Passaging Process of Mouse Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are a promising cell source because of their pluripotency and self-renewal abilities. However, there is a risk of pluripotency loss during cell expansion. Particularly, cell passaging is associated with a higher risk of decreasing cell quality. There are two iPSC passaging methods: single-cell and clump passaging. Single-cell passaging is a rapid and simple method for cell manipulation, whereas clump passaging is superior for maintaining iPSC pluripotency. Therefore, clump passaging is a robust method for expanding iPSCs while maintaining their pluripotency. However, clump size control during clump passaging is difficult because colony fragmentation is performed manually by pipetting the colonies detached from the cell culture substrates. In this study, the effect of pipetting on iPSC colony fragmentation was evaluated and the relationship between iPSC clump size and pluripotency was clarified. An automated pipetting device was developed to standardize the clump passage process. The effect of clump size on the pluripotency and proliferative capacity of mouse iPSCs was investigated. Clump size was controlled by varying the number of pipetting cycles, and pluripotency and proliferation were assessed via alkaline phosphatase staining and flow cytometry. Our results revealed that a decrease in clump size corresponded to an increase in cell proliferation, while pluripotency maintenance was optimized under specific clump sizes. These results underscore the significance of clump size for stem cell quality, emphasizing the need for a balanced approach to maintain pluripotency while fostering proliferation in the cell expansion culture for iPSCs.

Histological Analysis of Somatic Embryogenesis from Immature Zygotic Embryo of Wild Banana Musa acuminata ssp. malaccensis

Somatic embryogenesis, a crucial plant regeneration method, has become indispensable for crop improvement, particularly for species reliant on somatic cell manipulation techniques. Optimization of this process necessitates an understanding of the developmental stages involved. This study investigates the histological aspects of somatic embryogenesis in Musa acuminata ssp. malaccensis derived from immature zygotic embryos. Through detailed histological analysis, we aimed to elucidate the morphological changes and cellular organization occurring during the various stages of somatic embryogenesis, from induction, culture proliferation, and somatic embryo development to plantlet conversion. The initial stages of embryogenesis, characterized by nodules, were primarily composed of meristematic cells with high cell division activity. These cells contained tetrad-like structures that could develop into distinct two- and four-celled proembryoids or proembryogenic aggregates. Our histo-anatomical analysis revealed that embryogenic cultures proliferated through multiple pathways simultaneously: somatic embryo budding, proembryo formation, and pro-embryonic mass formation from both internal and peripheral cells. At the stage of somatic embryo development, embryos with a well-defined protoderm layer, containing cells with prominent nuclei and dense cytoplasm, potentially regenerate into plantlets. Furthermore, histological examination revealed the presence of procambium within mature somatic embryos, which subsequently developed into the vascular system of the complete plantlet

Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 (PKP2). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells' contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction-mediated diseases.

Identification and Manipulation of Spermatogonial Stem Cells with the Aim of Inducing Spermatogenesis in Vitro.

Assisted reproduction techniques for infertile men with non-obstructive azoospermia require a sufficient number of functional germ cells produced in vitro. Understanding the mechanisms that allow the resumption of spermatogenesis outside the testicular environment is crucial for fertility preservation in these patients. A review of the literature was conducted using databases such as PubMed, Scopus and Web of Science, with keywords including "spermatogonial stem cell," "germ cells," "male factor infertility," and "enrichment and propagation of SSCs in vitro." Currently, two models-"in vivo" and "in vitro"-have been developed for producing haploid germ cells. The "in vivo" models include spermatogonial stem cell transplantation and testicular xenograft techniques. In contrast, the "in vitro" models consist of conventional culture systems, organ culture, and three-dimensional culture systems, all designed to induce spermatogenesis in vitro. These culture systems enable the simulation of the seminiferous epithelium in vitro, which facilitates better regulation of cell-signaling pathways that control the self-renewal and differentiation of SSCs. This review provides up-to-date information on the organization of SSCs, focusing on the identification, proliferation, and differentiation of spermatogonia in vitro.

Controlling Cell Interactions with DNA Directed Assembly.

The creation of complex cellular environments is critical to mimicking tissue environments that will play a critical role in next-generation tissue engineering, stem cell programming, and therapeutic screening. To address this growing need, techniques capable of manipulating cell-cell and cell-material interactions are required that span single-cell to 3D tissue architectures. DNA programmed assembly and placement of cells present a powerful technique for the bottom-up synthesis of living microtissues for probing key questions in cell-cell and cell-material-driven behaviors through its refined control over placement and architecture. This review examines the current state of the art in the programming of cellular interactions with DNA and its applications spanning tissue model building, fundamental cellular biology, and cell manipulation for measurements across a host of applications.

DC voltage switching-based octuple-electrode microdevice for cell rotation and area-specific membrane capacitance measurement

Single-cell electrorotation plays an important role in the field of single-cell imaging and electric parameter measurement. However, reported cell rotation technology often adopts a quadruple-electrode structure and is excited by an AC signal. The distribution of electric field strength in the enclosed area is not uniform, and the rotation speed of the cells is related to the location in the area, so it is difficult to achieve uniformity of electric field distribution and the stationarity of rotation. This work proposes a DC voltage switching-based octuple-electrode microdevice for cell rotation and area-specific membrane capacitance measurements. This design can switch the DC voltages on each electrode periodically to produce a uniformly distributed rotating electric field. The rotation direction of the electric field can be realized by simply controlling the switching order of the analog switches. According to the theoretical single-cell model, the area-specific membrane capacitance of cells are determined through rotation movements. Simultaneously, based on simulation results, the rotation area is normalized to enhance the accuracy of the measuring electrical parameters. This study demonstrates the potential application of the proposed octuple-electrode DC voltage-based electro-rotation device for rapid, convenient, and cost-effective manipulation and electrical parameter measurement of single cells.

Manipulation of Cells Using Phenomena Induced by Electrostatic Field

Manipulation of Cells Using Phenomena Induced by Electrostatic Field

Cell Manipulation by Static Magnetic Field

Cell Manipulation by Static Magnetic Field

Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated sequence (CRISPR/Cas) system is a cutting-edge genome-editing tool employed to explore the functions of normal and disease-related genes. The CRISPR/Cas system has a remarkable diversity in the composition and architecture of genomic loci and Cas protein sequences. Owing to its excellent efficiency and specificity, this system adds an outstanding dimension to biomedical research on genetic manipulation of eukaryotic cells. However, safe, efficient, and specific delivery of this system to target cells and tissues and their off-target effects are considered critical bottlenecks for the therapeutic applications. Recently discovered anti-CRISPR proteins (Acr) play a significant role in limiting the effects of this system. Acrs are relatively small proteins that are highly specific to CRISPR variants and exhibit remarkable structural diversity. The in silico approaches, crystallography, and cryo-electron microscopy play significant roles in elucidating the mechanisms of action of Acrs. Acrs block the CRISPR/Cas system mainly by employing four mechanisms: CRISPR/Cas complex assembly interruption, target-binding interference, target cleavage prevention, and degradation of cyclic oligonucleotide signaling molecules. Engineered CRISPR/Cas systems are frequently used in gene therapy, diagnostics, and functional genomics. Understanding the molecular mechanisms underlying Acr action may help in the safe and effective use of CRISPR/Cas tools for genetic modification, particularly in the context of medicine. Thus, attempts to regulate prokaryotic CRISPR/Cas surveillance complexes will advance the development of antimicrobial drugs and treatment of human diseases. In this review, recent updates on CRISPR/Cas systems, especially CRISPR/Cas9 and Acrs, and their novel mechanistic insights are elaborated. In addition, the role of Acrs in the novel applications of CRISPP/Cas biotechnology for precise genome editing and other applications is discussed.

Spiral microchannels with concave cross-section for enhanced cancer cell inertial separation.

Inertial microfluidic technologies have proven effective for particle focusing and separation in many microchannels, typically the channels with the rectangular and trapezoidal shapes. To advance particle focusing in complex channels, we propose a spiral channel combining rectangular and concave cross-sections for high-resolution particle and cell focusing and separation. Numerical simulations were conducted to illustrate the effects of channel geometry on secondary flow distribution and particle focusing positions. The simulation shows the concave cross-section generates two asymmetrical Dean vortices skewing towards the inner and outer channel walls, resulting to stronger flow velocity magnitudes near the walls than the channel center. Consequently, larger particles focus near the inner wall, while smaller particles are trapped closer to the outer wall under the influence of the stronger velocity magnitude near the walls. A microfluidic chip with the proposed channelgeometry, along with a traditional rectangular channel, was fabricated by 3D printing and PDMS casting. Fluorescent microbeads were used to investigate inertial focusing and separation behaviors in the microfluidicchips. Experimental results show that the concave channel facilitates particle focusing or trapping much closer to the walls than the traditional rectangular channel, achieving better separation resolution. Finally, the proposed channel was applied to separate lung cancer A549 cells from human blood, achieving a cancer cell recovery rate of ~ 84.78% (enrichment ratio over 820-fold) and a blood cell rejection rate of ~ 99.88%. This innovative channel design in inertial microfluidics offers new insights for enhanced particle focusing and holds significant promise for cell manipulation with improved separation resolution.

A heterotypic tumor-on-a-chip platform for user-friendly combinatorial chemotherapeutic testing

BackgroundThree-dimensional (3D) tumor microdevices are promising platform for biomimetic antitumor prediction and high-throughput chemotherapeutic screening and play crucial roles in the exploration of cancer-associated pharmaceutics and therapeutics. Traditional cell manipulation tools (e.g., non-adhesive surfaces and hanging drops) and recent microengineered systems (e.g., microfluidic chips and micropatterned array chips) have progressed in terms of microscale control, substantial tumor production, programmable drug combinations, and throughput analysis. However, establishing a facile 3D tumor microdevice to construct heterotypic tumor-microenvironmental profiles and for throughput, implementable, multi-instrument-compatible analysis of chemotherapies to advance consumer-grade tumor modelling tools is still being explored. ResultsIn this study, we present a facilely operated tumor-on-a-chip platform for massive production of heterotypic 3D tumors and diverse investigations of combinatorial chemotherapy screening. Large quantity of heterotypic tumor generation with high geometric controllability (size difference: 19.6 μm) and operational repeatability (n = 10) was achieved using simple-to-fabricate micropatterned chips. Multiple characteristics of solid tumors, including phenotypic gradients (viability and proliferation) and heterogeneous cellular compositions (multi-cell participation and stroma composition), were reproduced in heterotypic tumors, being more biomimetic than homotypic tumors. We completed the user-friendly analytical evaluation of individual and combinatorial drug therapies, and demonstrated the high applicability of the platform in biomimetic tumor-related large-scale manipulation and on-chip analysis, as well as its high compatibility for off-chip detection. The entire operative process during tumor production and chemotherapy only requires the routine and easy-to-master pipetting manipulation. SignificanceThe establishment of a biomimetic and easy-to-use 3D tumor platform and the large-scale screening-like evaluation of combinatorial chemotherapies based on the usage of the micropatterned chip was achieved in a user-friendly manner. This advancement has significant application potential in the fields of oncology, drug discovery, and tissue engineering, and is expected to be valuable for developing accessible and generalizable tumor-on-a-chip microsystems for exploring cancer therapies.

Stimulation of an entorhinal-hippocampal extinction circuit facilitates fear extinction in a post-traumatic stress disorder model.

Effective psychotherapy of post-traumatic stress disorder (PTSD) remains challenging due to the fragile nature of fear extinction, for which ventral hippocampal CA1 (vCA1) region is considered as a central hub. However, neither the core pathway nor the cellular mechanisms involved in implementing extinction are known. Here, we unveil a direct pathway, where layer 2a fan cells in the lateral entorhinal cortex (LEC) target parvalbumin-expressing interneurons (PV-INs) in the vCA1 region to propel low gamma-band synchronization of the LEC-vCA1 activity during extinction learning. Bidirectional manipulations of either hippocampal PV-INs or LEC fan cells sufficed fear extinction. Gamma entrainment of vCA1 by deep brain stimulation (DBS) or noninvasive transcranial alternating current stimulation (tACS) of LEC persistently enhanced the PV-IN activity in vCA1, thereby promoting fear extinction. These results demonstrate that the LEC-vCA1 pathway forms a top-down motif to empower low gamma-band oscillations that facilitate fear extinction. Finally, application of low gamma DBS and tACS to a mouse model with persistent PTSD showed potent efficacy, suggesting that the dedicated LEC-vCA1 pathway can be stimulated for therapy to remove traumatic memory trace.

CRISPRoff epigenetic editing for programmable gene silencing in human cells without DNA breaks.

The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.

Giant pyramidal neurons of the primary motor cortex express vasoactive intestinal polypeptide (VIP), a known marker of cortical interneurons

Vasoactive intestinal polypeptide (VIP) is known to be present in a subclass of cortical interneurons. Here, using three different antibodies, we demonstrate that VIP is also present in the giant layer 5 pyramidal (Betz) neurons which are characteristic of the limb and axial representations of the marmoset primary motor cortex (cytoarchitectural area 4ab). No VIP staining was observed in smaller layer 5 pyramidal cells present in the primary motor facial representation (cytoarchitectural area 4c), or in the premotor cortex (e.g. the caudal subdivision of the dorsal premotor cortex, A6DC), indicating the selective expression of VIP in Betz cells. VIP in Betz cells was colocalized with neuronal specific marker (NeuN) and a calcium-binding protein parvalbumin (PV). PV also intensely labelled axon terminals surrounding Betz cell somata. VIP-positive interneurons were more abundant in the superficial cortical layers and constituted about 5–7% of total cortical neurons, with the highest density observed in area 4c. Our results demonstrate the expression of VIP in the largest excitatory neurons of the primate cortex, which may offer new functional insights into the role of VIP in the brain, and provide opportunities for genetic manipulation of Betz cells.

An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.