scholarly journals Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

2016 ◽  
Vol 8 (10) ◽  
pp. 63
Author(s):  
Saisai Wang ◽  
Yali Wang ◽  
Dan Shen ◽  
Li Zhang ◽  
Songlei Xue ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Transfer Efficiency ◽  
Transgenic Chicken ◽  
Simple Method ◽  
Gene Transfer Efficiency ◽  
Transgenic Chickens

<p>Transposon mediated transfection is a promising, safe, and convenient way to generate transgenic chicken compared with virus-mediated technology and the in vitro modification of primordial germ cells (PGCs). To establish a simple method for in vivo transfection of chicken PGCs, we applied four different transposon systems (PB, SB, Tol2, and ZB) to investigate the gene transfer efficiency of chicken gonads via direct injection of a mixture of transposon and transposase plasmids and transfection reagent (polyethylenimine, PEI) into the subgerminal cavity of Hamburger and Hamilton stage 2-3 chick embryos. We also compared the effect of the amount of plasmids injected on the gene transfer efficiency of chicken gonads. We found that over 70% of the gonads were green fluorescent protein (GFP)-positive across all four transposon groups, and that the proportion of GFP-positive gonads was not significantly different between different transposons. Some GFP positive cells in gonads were confirmed as germ cells by co-labeling with the germ cell specific antibody. We also found that the proportions of GFP-positive gonads decreased significantly with a decrease of plasmid dose from 100 ng to 20 or 50 ng. Here we revealed that a combination of transposons with PEI is a simple and efficient method for gene transfer into chicken gonads and able to transfect PGCs in vivo that could be used for the production of transgenic chickens.</p>

scholarly journals Guanidinylated block copolymers for gene transfer: A comparison with amine-based materials for in vitro and in vivo gene transfer efficiency

Biomaterials ◽  
2015 ◽  
Vol 54 ◽  
pp. 87-96 ◽  
Author(s):  
Jennifer L. Choi ◽  
James-Kevin Y. Tan ◽  
Drew L. Sellers ◽  
Hua Wei ◽  
Philip J. Horner ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Block Copolymers ◽  
Transfer Efficiency ◽  
Gene Transfer Efficiency ◽  
In Vivo Gene Transfer

scholarly journals Improvement in Adenoviral Gene Transfer Efficiency after Preincubation at +37°C in Vitro and in Vivo

2002 ◽  
Vol 5 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Maija Kossila ◽  
Suvi Jauhiainen ◽  
Mikko O. Laukkanen ◽  
Pauliina Lehtolainen ◽  
Maiju Jääskeläinen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transfer Efficiency ◽  
Adenoviral Gene Transfer ◽  
Gene Transfer Efficiency

scholarly journals Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Zihou Liu ◽  
Kristin Klose ◽  
Sebastian Neuber ◽  
Meng Jiang ◽  
Manfred Gossen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescent Protein ◽  
Left Ventricular ◽  
Transfer Efficiency ◽  
Slice Culture ◽  
Cell Tropism ◽  
Tissue Slices ◽  
Acetoxymethyl Ester ◽  
Gene Transfer Efficiency

Abstract Background Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. Methods LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. Results Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. Conclusions We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases.

scholarly journals Glycocalyx Restricts Adenoviral Vector Access to Apical Receptors Expressed on Respiratory Epithelium In Vitro and In Vivo: Role for Tethered Mucins as Barriers to Lumenal Infection

2004 ◽  
Vol 78 (24) ◽  
pp. 13755-13768 ◽  
Author(s):  
Jaclyn R. Stonebraker ◽  
Danielle Wagner ◽  
Robert W. Lefensty ◽  
Kimberlie Burns ◽  
Sandra J. Gendler ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgenic Mice ◽  
Airway Epithelium ◽  
Adenoviral Vector ◽  
Respiratory Epithelium ◽  
Transfer Efficiency ◽  
Gene Transfer Efficiency ◽  
Transfer Strategies

ABSTRACT Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

scholarly journals Nonviral Gene Therapy for Cancer: A Review

Diseases ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 57 ◽  
Author(s):  
Chiaki Hidai ◽  
Hisataka Kitano
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Viral Vectors ◽  
Viral Vector ◽  
Transfer Efficiency ◽  
Nonviral Vectors ◽  
Apparent Lack ◽  
Gene Transfer Efficiency ◽  
Relative Safety

Although the development of effective viral vectors put gene therapy on the road to commercialization, nonviral vectors show promise for practical use because of their relative safety and lower cost. A significant barrier to the use of nonviral vectors, however, is that they have not yet proven effective. This apparent lack of interest can be attributed to the problem of the low gene transfer efficiency associated with nonviral vectors. The efficiency of gene transfer via nonviral vectors has been reported to be 1/10th to 1/1000th that of viral vectors. Despite the fact that new gene transfer methods and nonviral vectors have been developed, no significant improvements in gene transfer efficiency have been achieved. Nevertheless, some notable progress has been made. In this review, we discuss studies that report good results using nonviral vectors in vivo in animal models, with a particular focus on studies aimed at in vivo gene therapy to treat cancer, as this disease has attracted the interest of researchers developing nonviral vectors. We describe the conditions in which nonviral vectors work more efficiently for gene therapy and discuss how the goals might differ for nonviral versus viral vector development and use.

Modification of a fiber protein in an adenovirus vector improves in vitro gene transfer efficiency to the mouse microglial cell line

2002 ◽  
Vol 324 (2) ◽  
pp. 145-148 ◽  
Author(s):  
Nobuhiko Omori ◽  
Hiroyuki Mizuguchi ◽  
Keiko Ohsawa ◽  
Shinichi Kohsaka ◽  
Takao Hayakawa ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Line ◽  
Microglial Cell ◽  
Adenovirus Vector ◽  
Transfer Efficiency ◽  
Gene Transfer Efficiency ◽  
Fiber Protein ◽  
Microglial Cell Line

scholarly journals Limited Entry of Adenovirus Vectors into Well-Differentiated Airway Epithelium Is Responsible for Inefficient Gene Transfer

1998 ◽  
Vol 72 (7) ◽  
pp. 6014-6023 ◽  
Author(s):  
Raymond J. Pickles ◽  
Douglas McCarty ◽  
Hirotoshi Matsui ◽  
Pádraig J. Hart ◽  
Scott H. Randell ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Adenovirus Vector ◽  
Airway Epithelial Cells ◽  
Transfer Efficiency ◽  
Apical Plasma Membrane ◽  
Airway Epithelial ◽  
Gene Transfer Efficiency ◽  
Well Differentiated

ABSTRACT Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of αvβ3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.

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