Tracking fluorescent protein transgenic cells using a robot microscope

2019 ◽  
Author(s):  
Toshiki Nozawa ◽  
Koichi Hashimoto
Keyword(s):  
Fluorescent Protein ◽  
Transgenic Cells

86 EFFECT OF HDAC2 INTERFERENCES ON HISTONE MODIFICATIONS IN MOUSE EARLY EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 191
Author(s):  
Q. Shen ◽  
Y. Liu ◽  
F. Yin ◽  
L. Yang ◽  
G. Li
Keyword(s):  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Fluorescent Protein ◽  
Basic Research ◽  
Negative Control ◽  
Immunofluorescent Staining ◽  
Control Group ◽  
Real Time Quantitative Pcr ◽  
Early Embryos ◽  
Transgenic Cells

One of the histone deacetylase members, HDAC2 plays an important role in chromatin remodeling and transcriptional repression. The present study was designed to improve histone acetylations by knocking down the expression of histone deacetylase with RNA interferences. According to the published mouse HDAC2 mRNA sequence (NM_008229.2, GI:87162463), as well as shRNA design principles, 3 interference fragments of 107 to 126 bps, 879 to 898 bps, and 1240 to 1259 bps and a negative control sequence were designed and synthesized, respectively. Four interference vectors expressing a red fluorescent protein were successfully constructed, which were named pCDsRed2-shRNA107, pCDsRed2-shRNA879, pCDsRed2-shRNA1240, and pCDsRed2-shRNAcontrol, respectively. These vectors were then transfected respectively to mouse fetal fibroblast cells. Real-time, quantitative PCR of the transgenic cells showed that the vectors mentioned above resulted in 0.81, 0.73, 0.16, and 0.80 times knockdown of hdac2 expression when compared with the nontransfected cells, which suggested that the piece of 1240 to 1259 bp was the most effective RNAi region. Immunofluorescent staining of the transgenic cells showed that the histone acetylations and methylations of H4K5ac, H3K9ac, H3K9me2, H3K27me3, and H3K4me3 were significantly higher in all of the interference vectors than they were in the negative control. Vector pCDsRed2-shRNA1240 was the most effective RNAi. After injection of pCDsRed2-shRNA1240 into zygote pronuclei, embryos developed to 2-cell, 8-cell embryos and blastocysts decreased to 96.6, 85.3 (P < 0.05), and 35.6 (P < 0.01) of the control group, respectively. High levels of H3K9ac, H4K5ac, H3K4me3, and H3K27me3 were observed in the RNAi embryos when compared with the controls. These results indicated that the knockdown of hdac2 by RNAi decreased the expression of HDAC2 and induced high expression of histone acetylations in both somatic cells and early embryos. This work was supported by the national basic research program of china (No. 2012CB22306).

330 TRICHOSTATIN A IMPROVES THE IN VITRO DEVELOPMENT OF CLONED BOVINE EMBRYOS RECONSTRUCTED WITH TRANSGENIC DONOR CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 313
Author(s):  
L. S. A. Camargo ◽  
R. J. Otero Arroyo ◽  
T. D. Araujo ◽  
G. N. Quinelato ◽  
C. R. C. Quintao ◽  
...  
Keyword(s):  
Embryo Development ◽  
Fluorescent Protein ◽  
Trichostatin A ◽  
Cell Lineage ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Donor Cells ◽  
Transgenic Cells ◽  
Cells Cultured

Trichostatin A (TSA), a histone deacetylase inhibitor, has been described as a potential modulator of nuclear reprogramming in bovine zygotes reconstructed by somatic cell nuclear transfer (SCNT), but with controversial results (Lee et al. 2011 J. Reprod. Dev. 57, 34–42; Sangalli et al. 2012 Cell Reprogramming 14, 1–13). The effect of TSA in zygotes reconstructed with transgenic cells cultured for long periods is not known. This study aimed to evaluate the effect of TSA on development of bovine embryos reconstructed with donor cells transfected with a green fluorescent protein (GFP)-reporter transgene. Bovine fibroblasts at second passage were transfected with lentiviral vectors carrying the GFP transgene and cultured at 37.5°C under 5% CO2 in air. Transfected cells were cultured for additional 10 passages to establish a cell lineage expressing the protein. In the 12th passage, the cells were frozen in 10% dimethyl sulfoxide plus FCS (Nutricell, Campinas, Brazil) and frozen–thawed cells expressing GFP were used as nucleus donors. In vitro-matured oocytes were enucleated, fused to GFP positive fibroblasts, and activated with ionomycin. Putative zygotes were randomly distributed into 2 groups: SCNT-CONT (n = 55): zygotes were cultured for 4 h in CR2aa medium plus BSA with 6-DMAP followed by 7 h in CR2aa medium plus 2.5% FCS; SCNT-TSA (n = 49): zygotes were cultured in the same conditions described above, but supplemented with 50 nM TSA (Sigma-Aldrich, St Louis, MO). Then, embryos from all groups were cultured in CR2aa supplemented with 2.5% FCS under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Evaluations of cleavage and blastocyst percentages were performed at 72 and 168 h post-activation, respectively, and 4 replicates were carried out. Expression of GFP in embryos at blastocyst stage was visualised using an epifluorescence microscope. Statistical analysis was performed by ANOVA and data are shown as mean ± SEM. No difference (P > 0.05) on cleavage percentage was found between groups (72.9 ± 11.3% and 66.1 ± 14.4% for SCNT-CONT and SCNT-TSA, respectively). The blastocyst percentage calculated based on putative zygotes tended (P = 0.077) to be higher for SCNT-TSA (16.7 ± 4.0%) than for SCNT-CONT (6.8 ± 2.3%). When the blastocyst percentage was calculated based on cleaved embryos, a higher rate (P < 0.05) was achieved in SCNT-TSA (26.7 ± 3.8%) than in SCNT-CONT (10.3 ± 3.6%) group. Blastocysts of both groups expressed GFP, with no difference among embryos. In a previous study, we reported that TSA had no positive effect on in vitro embryo development or gene expression, despite the reduction on apoptosis index [Camargo et al. 2011 Acta Sci. Vet. 39(Suppl.), S442; Camargo et al. 2012 Reprod. Fert. Dev. 24, 121–122). In the present study, however, the treatment with TSA of zygotes reconstructed with transgenic cells cultured for a long time improved embryo development without impairing GFP expression. This result suggests that TSA may be effective in clones reconstructed with transgenic cells. Supported by Embrapa 01.07.01.002, CBAB/CNPq, CAPES and Fapemig.

332 INDUCIBLE RED FLUORESCENT PROTEIN (RFP) EXPRESSION IN PORCINE FIBROBLASTS AND TRANSGENIC CLONED EMBRYOS USING piggyBac TRANSPOSITION

2011 ◽  
Vol 23 (1) ◽  
pp. 262 ◽  
Author(s):  
S. J. Kim ◽  
O. J. Koo ◽  
S. J. Park ◽  
J. H. Moon ◽  
D. K. Kwon ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Transgenic Pigs ◽  
Donor Cells ◽  
Transgenic Cells ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Animal Resources ◽  
Retrovirus Infection ◽  
Transposase Expression

Transgenic pigs are promising animal resources for human disease models and organ donors for xenotransplantation, because they resemble humans anatomically and physiologically. Transgenic pigs have been produced from transfected donor cells using several gene delivery systems including retrovirus infection. Recently, it has been reported that piggyBac (PB) transposition is a highly efficient tool in producing transgenic mice. This study investigated the use of PB transposition to establish transgenic cells and produce transgenic cloned embryos in pigs. We constructed plasmid DNA with red fluorescence protein (RFP) expressed by tetracycline-dependent cassette (from Addgene) with PB site using gateway cloning. We co-transfected porcine fibroblasts with the structured plasmid vector (pB-TET-DsRed), pB-rtTA (from Addgene), and a transposase expression vector pCy43 (Sanger Insitute, Hinxton, UK) using Fugene HD. After 24 h, 2 μg mL–1 doxycycline was added to the culture medium to turn on RFP expression. After 48 h of culture, 1 mg mL–1 neomycin was added to select stable RFP transfectants. Selected fibroblasts were cultured for 9 days without doxycycline, thus reducing RFP expression. After establishment of inducible RFP-expressing cells, the cells were used for somatic cell nuclear transfer. Embryos were cultured in porcine zygote medium-3, and 2 μg mL–1 doxycycline was added 5 days later. As a result, RFP expression was detected in the blastocysts. In conclusion, this study demonstrated that the inducible RFP gene in porcine fibroblasts and embryos was controlled by PB transposition system. Furthermore, this system could be a means of delivering an exogenous gene into porcine somatic cells and embryos for transgenic research. This study was supported by grants from MKE (#2009-67-10033839, #2009-67-10033805), IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), and BK21 program.

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 67-72 ◽  
Author(s):  
So Gun Hong ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
Min Jung Kim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Transfected Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

Construction of Ipr1 expression vector and development of cloned embryos in vitro

Zygote ◽  
2011 ◽  
Vol 21 (3) ◽  
pp. 265-269 ◽  
Author(s):  
Yongli Song ◽  
Xiaoning He ◽  
Song Hua ◽  
Jie Lan ◽  
Yonggang Liu ◽  
...  
Keyword(s):  
Expression Vector ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Pathogen Resistance ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells ◽  
Green Fluorescent ◽  
Bovine Oocytes

SummaryThe purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP–EGFP–Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light &gt;600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

Site-Specific Protein Covalent Attachment to Nanotubes and Its Electronic Impact on Single Molecule Function

2019 ◽  
Author(s):  
Adam Beachey ◽  
Harley Worthy ◽  
William David Jamieson ◽  
Suzanne Thomas ◽  
Benjamin Bowen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Functional Integration ◽  
Attachment Site ◽  
Covalent Attachment ◽  
Great Promise ◽  
Functional Coupling ◽  
Phenyl Azide ◽  
Electronic Impact ◽  
Protein Attachment

<p>Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.</p>

Sign in / Sign up

Export Citation Format

Share Document