scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

scholarly journals Engineering Viral Vectors for Acoustically Targeted Gene Delivery

2021 ◽  
Author(s):  
Hongyi Li ◽  
John E Heath ◽  
James S Trippett ◽  
Mikhail G. Shapiro ◽  
Jerzy O Szablowski
Keyword(s):  
Gene Delivery ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Systemic Circulation ◽  
Brain Regions ◽  
Transduction Efficiency ◽  
Site Specific ◽  
Targeted Gene Delivery ◽  
The Brain

Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, limiting their scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency, requires ultrasound parameters close to tissue damage limits, and results in undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.

Comparison of Viral Vectors for Gene Transfer into CLL Cells: Efficient Transduction with Adeno-Associated Virus-8 (AAV-8).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2985-2985 ◽  
Author(s):  
Andrew P. Jewell ◽  
Melanie Cochrane ◽  
Jenny McIntosh ◽  
Reuben Benjamin ◽  
Amit Nathwani
Keyword(s):  
Gene Transfer ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Dependent Manner ◽  
Gfp Expression ◽  
Cmv Promoter ◽  
Adeno Associated Virus ◽  
Green Fluorescent

Abstract Chronic Lymphocytic Leukaemia (CLL) remains largely incurable despite recent advances in therapy, and therefore alternative strategies are of interest in treating this disease. One such alternative is the use of gene therapy, but this relies on developing efficient gene transfer technologies. We have compared several viral vectors coding for green fluorescent protein (GFP) for their ability to transduce CLL cells. Three serotypes of adeno-associated virus (AAV) were used, AAV-2, AAV-5 and a relatively new isolate AAV-8, an EI-EIII deleted adenoviral 5 based vector, AV-5, all with GFP regulated by the CMV promoter, and a VSVG pseudotyped lentiviral vector in which GFP expression is controlled by EF1a promotor/enhancer complex. AV-5 resulted in variable GFP expression, 24.1±3.4%, n=10 but caused cell death at high multiplicities of infection (MOI). The lentiviral vector resulted in GFP expression of 23.5±2.6%, n=12, at the highest titre used, and expression declined in a distinct dose-dependent manner as titres were reduced. Of the AAV vectors, AAV-8 was the most efficient with GFP expression at 41.3±1.0% n=14. We conclude that AAV-8 is a promising viral vector for efficient transduction of CLL cells. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions.

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

2013 ◽  
Vol 95 (3) ◽  
pp. 319-329
Author(s):  
Atsushi Hirao ◽  
Tatsuo Kawarasaki ◽  
Kenjiro Konno ◽  
Satoko Enya ◽  
Masatoshi Shibata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Olfactory Epithelium ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Gfp Expression ◽  
Green Fluorescent

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

381 PRODUCTION OF A TRANSGENIC PIGLET BY A NEW SPERM INJECTION TECHNIQUE

2006 ◽  
Vol 18 (2) ◽  
pp. 297
Author(s):  
H. Y. Yong ◽  
C. Murphy ◽  
A. Rieke ◽  
L. Lai ◽  
Y. Hao ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Fluorescent Protein ◽  
Injection Technique ◽  
Motile Sperm ◽  
Gfp Expression ◽  
Injection Process ◽  
Development In Vitro ◽  
Green Fluorescent ◽  
Triton X

The technique for intracytoplasmic sperm injection (ICSI) has, until now, focused on scoring the tail of the sperm prior to catching and aspiration into the injection pipette. This is in spite of the fact that damage to the head would more closely simulate what occurs during normal fertilization. In addition, to aid in visualizing the injection process so that a reduced volume can be injected, the oocyte is generally centrifuged to clear a portion of the cytoplasm. Thus, with conventional ICSI, the sperm are immobilized with polyvinylpyrrolidone, repeatedly frozen and thawed, treated with DTT or Triton X-100, and severed between the head and tail; the oocyte is centrifuged or activated. All of the above treatments are designed to compensate for the intrinsic defects in conventional ICSI. Our objective was to use a modified ICSI procedure whereby aggressively motile sperm were captured onto the broken tip of an injection pipette and then injected into noncentrifuged oocytes. Damage to the head of the sperm occurred on the pipette or while pushed through the zona pellucida. These procedures are based on the work of Yong et al. 2003 Hum. Reprod. 18, 2390, where they achieved an improvement in development in vitro as compared to conventional methods. Ovaries were collected from prepubertal gilts, and oocytes were aspirated and matured in vitro. Sperm were collected from a transgenic boar carrying the green fluorescent protein (GFP) and frozen. After thawing, aggressively motile sperm were captured and injected through the zona pellucida and into the cytoplasm of the in vitro-matured oocytes. A total of 452 injected oocytes (43-171 oocytes per recipient) were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. GFP expression was observed in the nose and hooves by direct epifluorescent examination of the newborn piglet. This pattern of GFP expression is identical to that in non-ICSI-derived GFP pigs in this line. This result showed for the first time that this new sperm injection technique could be used for production of a viable transgenic piglet using in vitro-matured oocytes and frozen-thawed sperm.

Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent-protein genes

2020 ◽  
Author(s):  
Noriyuki Satoh ◽  
Koji Kinjo ◽  
Kohei Shintaku ◽  
Daisuke Kezuka ◽  
Hiroo Ishimori ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Biological Significance ◽  
Gene Expression Profiles ◽  
Gfp Expression ◽  
Red Fluorescent Proteins ◽  
Color Morphs ◽  
Acropora Tenuis ◽  
Green Fluorescent

ABSTRACTCorals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow-green (G) and purple (P) forms. The tips of axial coral polyps exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, the N and P morphs experienced bleaching, in which some N morphs died while P morphs recovered. In contrast, G morphs successfully withstood the stress. Symbiotic dinoflagellates are essential symbiotic partners of scleractinian corals. Photosynthetic activity of symbionts was reduced in July in N and P morphs; however, the three color-morphs host similar sets of Clade-C zoothanthellae, suggesting that beaching of N and P morphs cannot be attributed to differences in symbiont clades. The decoded Acropora tenuis genome includes five genes for green fluorescent proteins (GFP), two for cyan fluorescent proteins (CFP), three for red fluorescent proteins (RFP), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles demonstrated that (a) expression of CFP and REP was quite low in all three morphs, (b) P morphs expressed higher levels of ChrP, (c) both N and G morphs expressed GFP highly, and (d) GFP expression was reduced in N morphs, compared to G morphs, which maintained higher levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of Acropora tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs.

scholarly journals An improved molecular tool for screening bacterial colonies using GFP expression enhanced by a Dictyostelium sequence

BioTechniques ◽  
2020 ◽  
Vol 68 (2) ◽  
pp. 91-95 ◽  
Author(s):  
Tomo Kondo ◽  
Shigehiko Yumura
Keyword(s):  
Escherichia Coli ◽  
Gene Sequence ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Magnetospirillum Gryphiswaldense ◽  
E Coli ◽  
Molecular Tool ◽  
Visual Confirmation ◽  
Green Fluorescent ◽  
User Friendly

During molecular cloning, screening bacterial transformants is a time-consuming and labor-intensive process; however, tractable tools that can be applied to various vectors for visual confirmation of desired colonies are limited. Recently, we reported that translational enhancement by a Dictyostelium gene sequence (TED) boosted protein expression even without an expression inducer in Escherichia coli. Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein enhanced by TED. By inserting a module related to TED into the cloning site in advance, we effectively screened E. coli colonies harboring the desired plasmid functions in a prokaryote ( Magnetospirillum gryphiswaldense) or eukaryote ( Dictyostelium discoideum). Thus, our system represents a user-friendly technique for cloning.

scholarly journals Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

2006 ◽  
Vol 72 (9) ◽  
pp. 6088-6093 ◽  
Author(s):  
Helen Rawsthorne ◽  
Kevin N. Turner ◽  
David A. Mills
Keyword(s):  
Fluorescent Protein ◽  
Group Ii Intron ◽  
Multicopy Plasmid ◽  
Bacterial Genomes ◽  
Gfp Expression ◽  
High Frequencies ◽  
Multicopy Integration ◽  
Group Ii ◽  
Green Fluorescent ◽  
High Level

ABSTRACT Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

scholarly journals In vivo analysis of key elements within the renin regulatory region

2008 ◽  
Vol 35 (3) ◽  
pp. 243-253 ◽  
Author(s):  
Sean T. Glenn ◽  
Craig A. Jones ◽  
Li Pan ◽  
Kenneth W. Gross
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Region ◽  
Renin Angiotensin System ◽  
Artificial Chromosome ◽  
Proximal Promoter ◽  
Gfp Expression ◽  
Renin Gene ◽  
Green Fluorescent

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

Nuclear localization of angiotensinogen in astrocytes

2005 ◽  
Vol 288 (2) ◽  
pp. R539-R546 ◽  
Author(s):  
Mikhiela Sherrod ◽  
Xuebo Liu ◽  
Xiaoji Zhang ◽  
Curt D. Sigmund
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Endocrine Function ◽  
Astrocytoma Cell ◽  
Protein Encoding ◽  
Localization Signal ◽  
Green Fluorescent ◽  
The Brain

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.

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