Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

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.

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Expression of Green Fluorescent Protein in Streptococcus gordonii DL1 and Its Use as a Species-Specific Marker in Coadhesion with Streptococcus oralis 34 in Saliva-Conditioned Biofilms In Vitro

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.4074-4083.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 4074-4083 ◽

Cited By ~ 35

Author(s):

Marcelo B. Aspiras ◽

Karen M. Kazmerzak ◽

Paul E. Kolenbrander ◽

Roderick McNab ◽

Neil Hardegen ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Specific Marker ◽

Streptococcus Gordonii ◽

Gfp Expression ◽

Streptococcus Oralis ◽

Oral Biofilms ◽

Species Specific ◽

Green Fluorescent

ABSTRACT Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter,PhppA, which is situated upstream of the chromosomalhppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp(′gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-′gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 fromPhppA and that S. gordonii DL1 transformed with the PhppA-′gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement ofS. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.

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

Scientific Reports ◽

10.1038/s41598-021-81046-5 ◽

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.

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Focal Distribution of Baculovirus IE1 Triggered by Its Binding to the hr DNA Elements

Journal of Virology ◽

10.1128/jvi.79.1.39-46.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 39-46 ◽

Cited By ~ 17

Author(s):

Toshihiro Nagamine ◽

Yu Kawasaki ◽

Tetsutaro Iizuka ◽

Shogo Matsumoto

Keyword(s):

Fluorescent Protein ◽

Direct Repeat ◽

Single Copy ◽

Targeted Mutagenesis ◽

Homologous Region ◽

Focus Formation ◽

Primary Determinant ◽

Dna Elements ◽

Species Specific ◽

Green Fluorescent

ABSTRACT In BmN cells infected with the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), IE1, a principal transcriptional activator, localizes to sites of viral DNA replication. IE1 initially displays focal distribution in BmNPV-infected cells prior to DNA synthesis, whereas the protein expressed by transfection with the ie1 gene is distributed throughout the nucleoplasm instead of localized to discrete subnuclear structures. To identify the inducer of focus formation for IE1, we conducted transfection experiments with an IE1-GFP construct and found that cotransfection with genomic DNA fragments bearing the homologous region (hr) sequences caused the formation of IE1-green fluorescent protein (GFP) foci. The transfection of insect cells with a single plasmid containing exclusively the hr3 sequence and the IE1-GFP gene was sufficient to form IE1-GFP foci. These results suggest that hr elements are a primary determinant of the focal distribution of IE1. An analysis of a series of hr3 deletion mutants showed that a single copy of the direct repeat could induce the formation of IE1 foci. Targeted mutagenesis within the hr-binding domain of IE1-GFP caused impairment of the hr-dependent IE1 localization, suggesting that binding of IE1 to the hr elements is essential for the onset of IE1 focus formation. The observation of BmNPV IE1 foci in non-BmNPV-susceptible cells suggests that no species-specific factors are required for hr-dependent IE1 focus formation.

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

Acta Zoologica ◽

10.1111/azo.12029 ◽

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

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Could Cells from Your Nose Fix Your Heart? Transplantation of Olfactory Stem Cells in a Rat Model of Cardiac Infarction

The Scientific World JOURNAL ◽

10.1100/tsw.2010.40 ◽

2010 ◽

Vol 10 ◽

pp. 422-433 ◽

Cited By ~ 11

Author(s):

Cameron McDonald ◽

Alan Mackay-Sim ◽

Denis Crane ◽

Wayne Murrell

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Fluorescent Protein ◽

Genetically Engineered ◽

Olfactory Mucosa ◽

Cell Transplant ◽

Adult Rat ◽

Cardiac Ventricle ◽

Green Fluorescent

This study examines the hypothesis that multipotent olfactory mucosal stem cells could provide a basis for the development of autologous cell transplant therapy for the treatment of heart attack. In humans, these cells are easily obtained by simple biopsy. Neural stem cells from the olfactory mucosa are multipotent, with the capacity to differentiate into developmental fates other than neurons and glia, with evidence of cardiomyocyte differentiationin vitroand after transplantation into the chick embryo. Olfactory stem cells were grown from rat olfactory mucosa. These cells are propagated as neurosphere cultures, similar to other neural stem cells. Olfactory neurospheres were grownin vitro, dissociated into single cell suspensions, and transplanted into the infarcted hearts of congeneic rats. Transplanted cells were genetically engineered to express green fluorescent protein (GFP) in order to allow them to be identified after transplantation. Functional assessment was attempted using echocardiography in three groups of rats: control, unoperated; infarct only; infarcted and transplanted. Transplantation of neurosphere-derived cells from adult rat olfactory mucosa appeared to restore heart rate with other trends towards improvement in other measures of ventricular function indicated. Importantly, donor-derived cells engrafted in the transplanted cardiac ventricle and expressed cardiac contractile proteins.

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A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

Analytical Biochemistry ◽

10.1016/j.ab.2005.02.006 ◽

2005 ◽

Vol 342 (2) ◽

pp. 341-344 ◽

Cited By ~ 16

Author(s):

Dineshkumar H. Dandekar ◽

Manish Kumar ◽

Jayashree S. Ladha ◽

Krishna N. Ganesh ◽

Debashis Mitra

Keyword(s):

Green Fluorescent Protein ◽

Quantitative Method ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Green Fluorescent

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Autophagy and the cvt Pathway Both Depend onAUT9

Journal of Bacteriology ◽

10.1128/jb.182.8.2125-2133.2000 ◽

2000 ◽

Vol 182 (8) ◽

pp. 2125-2133 ◽

Cited By ~ 67

Author(s):

Thomas Lang ◽

Steffen Reiche ◽

Michael Straub ◽

Monika Bredschneider ◽

Michael Thumm

Keyword(s):

Protein Turnover ◽

Protein Transport ◽

Fluorescent Protein ◽

Genomic Library ◽

Integral Membrane Protein ◽

Biologically Active ◽

Transport Pathways ◽

Rich Media ◽

Green Fluorescent ◽

Mutant Cells

ABSTRACT In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen foraut − mutants and isolated aut9-1cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic withCVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.

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Myofibroblast Development Is Characterized by Specific Cell-Cell Adherens Junctions

Molecular Biology of the Cell ◽

10.1091/mbc.e04-05-0386 ◽

2004 ◽

Vol 15 (9) ◽

pp. 4310-4320 ◽

Cited By ~ 138

Author(s):

B. Hinz ◽

P. Pittet ◽

J. Smith-Clerc ◽

C. Chaponnier ◽

J.-J. Meister

Keyword(s):

Fluorescent Protein ◽

Contractile Activity ◽

Adherens Junctions ◽

Smooth Muscle Actin ◽

Flow Chamber ◽

Dermal Fibroblasts ◽

Mechanical Resistance ◽

Specific Cell ◽

Collagen Gels ◽

Green Fluorescent

Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with α-smooth muscle actin (α-SMA)-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of α-SMA-positive and -negative fibroblasts in suspension. AJs of plated myofibroblasts are reinforced by α-SMA–mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits α-SMA–mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-α–catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity.

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

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab381 ◽

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.

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

10.1101/2020.08.28.272823 ◽

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.

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