Optimization of Cell-Based cDNA Microarray Conditions for Gene Functional Studies in HEK293 Cells

Since the cell-based cDNA microarray (CBCM) technique has been a useful tool for gain-of-function studies, many investigators have used CBCMs to identify interesting genes. However, this method requires better-established conditions to ensure high reverse transfection efficiency without cross-contamination. Therefore, we optimized CBCM techniques through various means. We determined that Lipofectamine 2000 was the most appropriate transfection reagent by evaluating eight commercialized reagents, and we determined that the most effective concentrations for printing solution constituents were 0.2 M sucrose (to yield a final concentration of 32 mM) and 0.2% gelatin (to yield a final concentration 0.075%). After examining various combinations, we also determined that the best concentrations of cDNA and transfection reagent for optimal reverse transfection efficiency were 1.5 µg/5 µL of cDNA and 5.5 µL of Lipofectamine 2000. Finally, via a time course, we determined that 72 h was the most effective reaction duration for reverse transfection, and we confirmed the stability of cDNA spot activity of CBCMs for various storage periods. In summary, the CBCM conditions that we have identified can provide more effective outcomes for cDNA reverse transfection on microarrays.

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Nanocarriers, Progenitor Cells, Combinational Approaches, and New Insights on the Retinal Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms22041776 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1776

Author(s):

Elham Pishavar ◽

Hongrong Luo ◽

Johanna Bolander ◽

Antony Atala ◽

Seeram Ramakrishna

Keyword(s):

Drug Delivery ◽

Progenitor Cells ◽

Transfection Efficiency ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Age Related Macular Degeneration ◽

Therapeutic Approaches ◽

Age Related ◽

Nanofibrous Scaffolds ◽

The Stability

Progenitor cells derived from the retinal pigment epithelium (RPECs) have shown promise as therapeutic approaches to degenerative retinal disorders including diabetic retinopathy, age-related macular degeneration and Stargardt disease. However, the degeneration of Bruch’s membrane (BM), the natural substrate for the RPE, has been identified as one of the major limitations for utilizing RPECs. This degeneration leads to decreased support, survival and integration of the transplanted RPECs. It has been proposed that the generation of organized structures of nanofibers, in an attempt to mimic the natural retinal extracellular matrix (ECM) and its unique characteristics, could be utilized to overcome these limitations. Furthermore, nanoparticles could be incorporated to provide a platform for improved drug delivery and sustained release of molecules over several months to years. In addition, the incorporation of tissue-specific genes and stem cells into the nanostructures increased the stability and enhanced transfection efficiency of gene/drug to the posterior segment of the eye. This review discusses available drug delivery systems and combination therapies together with challenges associated with each approach. As the last step, we discuss the application of nanofibrous scaffolds for the implantation of RPE progenitor cells with the aim to enhance cell adhesion and support a functionally polarized RPE monolayer.

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Calcium phosphate phase transition in the presence of Aminosilane

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409932x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C67-C67

Author(s):

Babak Mostaghaci ◽

Brigitta Loretz ◽

Robert Haberkorn ◽

Guido Kickelbick ◽

Claus-Michael Lehr

Keyword(s):

Phase Transition ◽

Calcium Phosphate ◽

Blood Compatibility ◽

Transfection Efficiency ◽

Hek293 Cells ◽

Step Method ◽

Calcium Phosphate Nanoparticles ◽

Amine Groups ◽

The Impact

Calcium phosphate has been the point of interest for in vitro gene delivery for many years because of its biocompatibility and straight forward application. However, there are some limitations regarding in vivo administration of these particles mostly because of vast agglomeration of the particles and lack of strong bond between the particles and pDNA. We introduced a simple single step method to functionalize calcium phosphate nanoparticles with Aminosilanes having a different number of amine groups. The nanoparticles were characterized chemically and structurally and their toxicity and interaction with pDNA were studied as well. Results revealed that different crystalline phase of calcium phosphate nanoparticles (Brushite and Hydroxyapatite) with a size below 150 nm were prepared, depending on conditions of synthesis and phase, each with a narrow size distribution. The aminosilane agents caused oriented nucleation and growth of crystallites and can decrease the pH for producing hydroxyapatite phase. The phenomenon could be revealed with the presence of anisotropy in the structure of synthesized hydroxyapatite. The number of amine groups in the Aminosilane agent could change the phase transition pH. Brushite particles revealed to have stronger interaction with pDNA mostly because of their higher positive surface charge. Both particles showed blood compatibility and negligible toxicity. Transfection experiment revealed the capability of both brushite and hydroxyapatite particles to transfect A549 and HEK293 cells. The new modified nanoparticles can be stored in a dried state and re-dispersed easily at the time of administration. Moreover, the transfection efficiency is higher in comparison with conventional calcium phosphate. This study showed the impact of presence and type of the modifying agent on the crystal structure and the amount of surface functionalization of nanoparticles, which in consequence influenced their interaction with cells.

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Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector

Plants ◽

10.3390/plants10122636 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2636

Author(s):

Ganeshan Sivanandhan ◽

Solhee Bae ◽

Chaemin Sung ◽

Su Ryun Choi ◽

Geung-Joo Lee ◽

...

Keyword(s):

Chinese Cabbage ◽

Binary Vector ◽

Transfection Efficiency ◽

Genome Structure ◽

Transformation Method ◽

Protoplast Isolation ◽

Breeding Cycle ◽

Functional Studies ◽

Gfp Reporter Gene ◽

Protoplast Transfection

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

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Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

Applied and Environmental Microbiology ◽

10.1128/aem.02940-15 ◽

2015 ◽

Vol 82 (1) ◽

pp. 289-296 ◽

Cited By ~ 8

Author(s):

Varvara Tsilia ◽

Frederiek-Maarten Kerckhof ◽

Andreja Rajkovic ◽

Marc Heyndrickx ◽

Tom Van de Wiele

Keyword(s):

Bacillus Cereus ◽

Food Poisoning ◽

Intestinal Bacteria ◽

Low Ph ◽

Final Concentration ◽

Adhesion Assay ◽

Content Type ◽

The Stability ◽

Bacterial Concentrations

ABSTRACTAdhesion to the intestinal epithelium could constitute an essential mechanism ofBacillus cereuspathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacteriumB. cereusNVH 0500/00 was exposedin vitroto gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherentB. cereusafter simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated.B. cereuscould grow and simultaneously adhere to mucin duringin vitroileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration ofB. cereusin the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml−1. At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retainB. cereusand to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.

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Effects of Decomplexation Rates on Ternary Gene Complex Transfection with α-Poly(l-Lysine) or ε-Poly(l-Lysine) as a Decomplexation Controller in An Easy-To-Transfect Cell or A Hard-To-Transfect Cell

Pharmaceutics ◽

10.3390/pharmaceutics12060490 ◽

2020 ◽

Vol 12 (6) ◽

pp. 490

Author(s):

Kyoungnam Kim ◽

Kitae Ryu ◽

Hana Cho ◽

Min Suk Shim ◽

Yong-Yeon Cho ◽

...

Keyword(s):

Transfection Efficiency ◽

Tight Binding ◽

Cationic Polymer ◽

Hek293 Cells ◽

Primary Amines ◽

Gene Complex ◽

Zeta Potentials ◽

Branched Polyethylenimine ◽

Potential Factor ◽

Different Characteristics

The tight binding of pDNA with a cationic polymer is the crucial requirement that prevents DNA degradation from undesired DNase attack to safely deliver the pDNA to its target site. However, cationic polymer-mediated strong gene holding limits pDNA dissociation from the gene complex, resulting in a reduction in transfection efficiency. In this study, to control the decomplexation rate of pDNA from the gene complex in a hard-to-transfect cell or an easy-to-transfect cell, either α-poly(l-lysine) (APL) or ε-poly(l-lysine) (EPL) was incorporated into branched polyethylenimine (bPEI)-based nanocomplexes (NCs). Compared to bPEI/pDNA NCs, the addition of APL or EPL formed smaller bPEI-APL/pDNA NCs with similar zeta potentials or larger bPEI-EPL/pDNA NCs with reduced zeta potentials, respectively, due to the different characteristics of the primary amines in the two poly(l-lysine)s (PLs). Interestingly, although both bPEI-APL/pDNA NCs and bPEI-EPL/pDNA NCs showed similar pDNA compactness to bPEI/pDNA NCs, the addition of APL or EPL resulted in slower or faster pDNA release, respectively, from the bPEI-PL/pDNA NCs than from the bPEI/pDNA NCs. bPEI-EPL/pDNA NCs with a decomplexation enhancer (i.e., EPL) improved the transfection efficiency (TE) in both a hard-to-transfect HepG2 cell and an easy-to-transfect HEK293 cell. However, although a decomplexation inhibitor (i.e., APL) reduced the TE of bPEI-APL/pDNA NCs in both cells, the degree of reduction in the TE could be compensated by PL-mediated enhanced nuclear delivery, particularly in HepG2 cells but not HEK293 cells, because both PLs facilitate nuclear localization of the gene complex per its cellular uptake. In conclusion, a decomplexation rate controller could be a potential factor to establish a high TE and design clinically available gene complex systems.

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Discordant Temporal Turnovers of Sediment Bacterial and Eukaryotic Communities in Response to Dredging: Nonresilience and Functional Changes

Applied and Environmental Microbiology ◽

10.1128/aem.02526-16 ◽

2016 ◽

Vol 83 (1) ◽

Cited By ~ 6

Author(s):

Na Zhang ◽

Xian Xiao ◽

Meng Pei ◽

Xiang Liu ◽

Yuting Liang

Keyword(s):

Time Course ◽

Species Turnover ◽

Macrobenthic Community ◽

Ecosystem Functions ◽

Rrna Gene ◽

Functional Changes ◽

Macrobenthic Communities ◽

Temporal Turnover ◽

The Stability ◽

Ecosystem Multifunctionality

ABSTRACT To study the stability and succession of sediment microbial and macrobenthic communities in response to anthropogenic disturbance, a time-series sampling was conducted before, during, and 1 year after dredging in the Guan River in Changzhou, China, which was performed with cutter suction dredgers from 10 April to 20 May 2014. The microbial communities were analyzed by sequencing bacterial 16S rRNA and eukaryotic 18S rRNA gene amplicons with Illumina MiSeq, and the macrobenthic community was identified using a morphological approach simultaneously. The results indicated that dredging disturbance significantly altered the composition and structures of sediment communities. The succession rates of communities were estimated by comparing the slopes of time-decay relationships. The temporal turnover of microeukaryotes (w = 0.3251, P < 0.001 [where w is a measure of the rate of log(species turnover) across log(time)]) was the highest, followed by that of bacteria (w = 0.2450, P < 0.001), and then macrobenthos (w = 0.1273, P < 0.001). During dredging, the alpha diversities of both bacterial and microeukaryotic communities were more resistant, but their beta diversities were less resistant than that of macrobenthos. After recovery for 1 year, all three sediment communities were not resilient and had reached an alternative state. The alterations in sediment community structure and stability resulted in functional changes in nitrogen and carbon cycling in sediments. Sediment pH, dissolved oxygen, redox potential, and temperature were the most important factors influencing the stability of sediment communities and ecosystem multifunctionality. This study suggests that discordant temporal turnovers and nonresilience of sediment communities under dredging resulted in functional changes, which are important for predicting sediment ecosystem functions under anthropogenic disturbances. IMPORTANCE Understanding the temporal turnover and stability of biotic communities is crucial for predicting the responses of sediment ecosystems to dredging disturbance. Most studies to date focused on the bacterial or macrobenthic community, only at two discontinuous time points, before and after dredging, and hence, it was difficult to analyze the community succession. This study first compared the stabilities and temporal changes of sediment bacterial, microeukaryotic, and macrobenthic communities at a continuous time course. The results showed that discordant responses of the three communities are mainly related to their different biological inherent attributes, and sensitivities to sediment geochemical variables change with dredging, resulting in changes in sediment ecosystem multifunctionality.

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Work Flow for Multiplexing siRNA Assays by Solid-Phase Reverse Transfection in Multiwell Plates

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108320133 ◽

2008 ◽

Vol 13 (7) ◽

pp. 575-580 ◽

Cited By ~ 56

Author(s):

Holger Erfle ◽

Beate Neumann ◽

Phill Rogers ◽

Jutta Bulkescher ◽

Jan Ellenberg ◽

...

Keyword(s):

Mammalian Cells ◽

Solid Phase ◽

Transfection Efficiency ◽

Rt Pcr ◽

Flexible Tool ◽

Cellular Assays ◽

Long Term Storage ◽

Cell Microarrays ◽

Reverse Transfection ◽

Multiwell Plates

Solid-phase reverse transfection on cell microarrays is a high-throughput method for the parallel transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as reverse transcriptase—polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase reverse transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The method retains all advantages of solid-phase reverse transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the “ready to transfect” multiwell plates, there is no reduction in their transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays. ( Journal of Biomolecular Screening. 2008:575-580)

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Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

Biochemical Journal ◽

10.1042/bj2300797 ◽

1985 ◽

Vol 230 (3) ◽

pp. 797-805 ◽

Cited By ~ 25

Author(s):

M G Tordi ◽

M C Silvestrini ◽

A Colosimo ◽

L Tuttobello ◽

M Brunori

Keyword(s):

Pseudomonas Aeruginosa ◽

Steady State ◽

Cytochrome C ◽

Cytochrome Oxidase ◽

Binding Sites ◽

Time Course ◽

Product Inhibition ◽

Inactive Form ◽

Catalytically Active ◽

The Stability

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a ‘half-hearted’ behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

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The Regulation of Natural Food Dyes Curcumin on the Amyloidogenic Pathway of APP

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.781-784.1160 ◽

2013 ◽

Vol 781-784 ◽

pp. 1160-1163

Author(s):

Xiong Zhang ◽

Jie Yun Sun ◽

Hong Mei Zhang ◽

Lu Si ◽

Yu Li

Keyword(s):

Time Course ◽

Mrna Level ◽

Time Dependent ◽

Hek293 Cells ◽

Natural Food ◽

Inhibition Effect ◽

Dose Time ◽

Protein Levels ◽

Food Dyes

As a promising prevention and therapeutic intervention in Alzheimer’s disease, natural food dyes curcumin could obviously inhibit the generation of Aβ, but the mechanism is not fully defined. This study aims to investigate the effects of curcumin on the amyloidogenic pathway of APP in vitro. Plasmids APPswe and BACE1-mychis were transiently co-transfected into SH-SY5Y and HEK293 cells. Then, they were treated with curcumin at 0, 1.25, 5, 20 μmol/L for 24 h, or with curcumin at 5μmol/L for 0, 12, 24 and 48 h for the time course assay. Our findings showed that curcumin could inhibit the expression of the APP at mRNA level; the expression of the BACE1 and C99 at mRNA and protein levels, furthermore it could inhibit the generation of Aβ40/42, and those changes were dose-time dependent (p<0.05). Our study indicates that Aβ40/42 generation inhibition effect of curcumin might due to its influence on amyloidogenic pathway. This may provide important experimental basis for AD treatment with curcumin.

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Peptide amphiphiles with multifunctional fragments promoting cellular uptake and endosomal escape as efficient gene vectors

Journal of Materials Chemistry B ◽

10.1039/c4tb01353k ◽

2015 ◽

Vol 3 (6) ◽

pp. 1068-1078 ◽

Cited By ~ 24

Author(s):

Liang Luan ◽

Qingbin Meng ◽

Liang Xu ◽

Zhao Meng ◽

Husheng Yan ◽

...

Keyword(s):

Gene Delivery ◽

Cellular Uptake ◽

Transfection Efficiency ◽

Endosomal Escape ◽

Peptide Amphiphiles ◽

Gene Delivery Vectors ◽

Efficient Gene ◽

Gene Vectors ◽

Lipofectamine 2000

A series of peptides containing multiple functional fragments were designed as gene-delivery vectors with transfection efficiency comparable to Lipofectamine 2000.

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