scholarly journals CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

2021 ◽  
Author(s):  
Enrique Lin Shiao ◽  
Wolfgang G Pfeifer ◽  
Brian R Shy ◽  
Mohammad Saffari Doost ◽  
Evelyn Chen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetic Material ◽  
Specific Cell ◽  
Dna Nanostructures ◽  
Primary Cells ◽  
Cell Nuclei ◽  
Genomic Integration ◽  
Dna Nanostructure ◽  
Human Primary Cells

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000's of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei.

In vivo performance of a dual genetic marker, manA-gfp, in transgenic bentgrass

Genome ◽  
2005 ◽  
Vol 48 (4) ◽  
pp. 722-730 ◽  
Author(s):  
Daolin Fu ◽  
Yanmei Xiao ◽  
Subbaratnam Muthukrishnan ◽  
George H Liang
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fluorescent Protein ◽  
Phosphomannose Isomerase ◽  
Genomic Integration ◽  
Chlorophenol Red ◽  
Marker Combination ◽  
Genes Encoding ◽  
Green Fluorescent ◽  
Fluorescence Stereomicroscope

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1 generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.Key words: bentgrass, Agrobacterium tumefaciens-mediated transformation, chlorophenol red assay, phosphomannose isomerase (PMI).

scholarly journals In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽  
2014 ◽  
Vol 147 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Franziska Schmerse ◽  
Katja Woidacki ◽  
Monika Riek-Burchardt ◽  
Peter Reichardt ◽  
Axel Roers ◽  
...  
Keyword(s):  
Mast Cells ◽  
Intravital Microscopy ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Specific Cell ◽  
Enhanced Yellow Fluorescent Protein ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

scholarly journals Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yilai Shu ◽  
Yong Tao ◽  
Wenyan Li ◽  
Jun Shen ◽  
Zhengmin Wang ◽  
...  
Keyword(s):  
Inner Ear ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Sensory Epithelium ◽  
Specific Cell ◽  
Supporting Cells ◽  
Auditory Hair Cells ◽  
A Genome ◽  
Adenovirus Vectors

Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction.

scholarly journals Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo

2009 ◽  
Vol 81 (8) ◽  
pp. 1403-1411 ◽  
Author(s):  
Aniuska Becerra ◽  
Rajas V. Warke ◽  
Katherine Martin ◽  
Kris Xhaja ◽  
Norma de Bosch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Primary Cells ◽  
Human Primary Cells

Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein

2012 ◽  
Vol 108 (8) ◽  
pp. 2323-2337 ◽  
Author(s):  
Walther Akemann ◽  
Hiroki Mutoh ◽  
Amélie Perron ◽  
Yun Kyung Park ◽  
Yuka Iwamoto ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Neural Circuit ◽  
Fluorescent Protein ◽  
Hippocampal Slices ◽  
Field Potential ◽  
Specific Cell ◽  
Fast Kinetics ◽  
Detection Range ◽  
Voltage Imaging

Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.

Conditional Reprogramming Inducing Clinical Cells Proliferation: New Research Tools in Tumor and Inflammatory-related Diseases

2020 ◽  
Vol 26 (22) ◽  
pp. 2657-2660 ◽  
Author(s):  
Jie chai ◽  
Li Han ◽  
Jianbo Zhang ◽  
Dali Han ◽  
Lei Zou ◽  
...  
Keyword(s):  
Cell Model ◽  
Culture Technique ◽  
Primary Cells ◽  
Preclinical Research ◽  
Human Primary Cells ◽  
Genetic Features ◽  
The Individual ◽  
New Research

In the era of precision medicine, establishing a patient-derived cell model is crucial, whether in vitro or in vivo. Compared to the traditional cell lines, patient-derived primary cells represent precise genetic features from specific patients, but poor proliferative activity of human primary cells restricts their popular application. Conditional reprogramming (CR) is a new cell culture technique to achieve rapid growth of patient-derived cells in vitro, making it possible to identify the individual difference and screen drugs sensitivity. In this review, we will summarize the application and limitation of CR in tumor and inflammatory-related diseases, indicating the prospect of this technique for preclinical research.

scholarly journals Gs-DREADD Knock-In Mice for Tissue-Specific, Temporal Stimulation of Cyclic AMP Signaling

2017 ◽  
Vol 37 (9) ◽  
Author(s):  
Dmitry Akhmedov ◽  
Maria G. Mendoza-Rodriguez ◽  
Kavitha Rajendran ◽  
Mario Rossi ◽  
Jürgen Wess ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Cre Recombinase ◽  
Designer Drugs ◽  
Camp Signaling ◽  
Luciferase Reporter ◽  
Specific Cell ◽  
Conditional Expression

ABSTRACT Hundreds of hormones and ligands stimulate cyclic AMP (cAMP) signaling in different tissues through the activation of G-protein-coupled receptors (GPCRs). Although the functions and individual effectors of cAMP signaling are well characterized in many tissues, pleiotropic effects of GPCR agonists limit investigations of physiological functions of cAMP signaling in individual cell types at different developmental stages in vivo. To facilitate studies of cAMP signaling in specific cell populations in vivo, we harnessed the power of DREADD (designer receptors exclusively activated by designer drugs) technology by creating ROSA26-based knock-in mice for the conditional expression of a Gs-coupled DREADD (rM3Ds-green fluorescent protein [GFP], or “GsD”). After Cre recombinase expression, GsD is activated temporally by the administration of the ligand clozapine N-oxide (CNO). In the same allele, we engineered a CREB-luciferase reporter transgene for noninvasive bioluminescence monitoring of CREB activity. After viral delivery of Cre recombinase to hepatocytes in vivo, GsD is expressed and allows CNO-dependent cAMP signaling and glycogen breakdown. The long-term expression of GsD in the liver results in constitutive CREB activity and hyperglycemia. ROSA26-Gs-DREADD mice can be used to study the physiological effects of cAMP signaling, acute or chronic, in liver or any tissue or cell type for which transgenic or viral Cre drivers are available.

scholarly journals Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1067
Author(s):  
Ting Li ◽  
Fengjiao Yao ◽  
Yacong An ◽  
Xundou Li ◽  
Jinhong Duan ◽  
...  
Keyword(s):  
Renal Clearance ◽  
Antitumor Efficacy ◽  
Holliday Junction ◽  
Dna Nanostructures ◽  
Dna Nanostructure ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Good Biocompatibility ◽  
Average Size

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.

Selective differentiation of central nervous system–derived stem cells in response to cues from specific regions of the developing brain

2007 ◽  
Vol 107 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Bonnie Vorasubin ◽  
John Weedin ◽  
Farid Saljooque ◽  
Nathan Wilkes ◽  
Hoi Sang U
Keyword(s):  
Stem Cells ◽  
Central Nervous System ◽  
Nervous System ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Genetically Engineered ◽  
Cell Contact ◽  
Specific Cell ◽  
Primary Cells ◽  
Protein Markers

Object Each region of the brain is distinguished by specific and distinct markers and functions. The authors hypothesized that each region possesses unique trophic properties that dictate and maintain its development. To test this hypothesis, they isolated central nervous system (CNS) stem cells from fetal rodents, and these rat CNS-derived stem cells (RSCs) were placed in coculture with primary cultures of the developing neonatal hippocampus and hypothalamus to determine whether region-specific primary cells would direct the differentiation of stem cells in a region-specific manner. Methods Primary cultures were first established from the neonatal (3–7 days postnatal) hippocampus and hypothalamus. Rodent CNS stem cells, which had been genetically engineered to express green fluorescent protein, were then placed in coculture with the primary CNS cells. The expression of region-specific markers in the RSCs was then evaluated after 2 weeks using immunocytochemistry. Data from previous studies have indicated that primary adult cells lack a differentiation-inducing capacity. Results When placed in coculture with primary CNS cells, RSCs began to express both neuronal (MAP2) and glial (glial fibrillary acidic protein) markers. Those that were placed in coculture with hippocampal cells expressed region-specific markers such as γ-aminobutyric acid, whereas those placed in coculture with hypothalamic cells expressed growth hormone–releasing hormone primarily in the hypothalamus. Conclusions Pluripotential RSCs were induced to express region-specific phenotypes on coculture with primary cells derived from the developing hippocampus and hypothalamus. The differentiation of RSCs into specific lineages on exposure to specific cell types is likely modulated through direct cell–cell contact. Secreted factors from the primary neural cells may also play a role in this induction. Such a differentiation influence is also likely age dependent.

scholarly journals In vivo cloning of artificial DNA nanostructures

2008 ◽  
Vol 105 (46) ◽  
pp. 17626-17631 ◽  
Author(s):  
Chenxiang Lin ◽  
Sherri Rinker ◽  
Xing Wang ◽  
Yan Liu ◽  
Nadrian C. Seeman ◽  
...  
Keyword(s):  
Dna Nanotechnology ◽  
Dna Nanostructures ◽  
Scientific Enterprise ◽  
Dna Nanostructure ◽  
Copy Numbers ◽  
Artificial Dna ◽  
Difficult Challenge ◽  
Endonuclease Vii

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.

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