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.

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.

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.

scholarly journals Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1386
Author(s):  
Jerzy Wiater ◽  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Daniel Lipiński
Keyword(s):  
Genetic Resource ◽  
Ex Vivo ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Fibroblast Cells ◽  
Control Groups ◽  
Transgenic Pigs ◽  
Cell Tissue ◽  
Donor Cells ◽  
Lower Expression

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA−) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA− cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA− hFUT2×hGLA cells as compared to the TSA− nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

Exploring Structural Dynamics and Optical Properties of UnaG Fluorescent Protein upon N57 Mutations

2021 ◽  
Author(s):  
Mohammad Asad ◽  
Adèle D Laurent
Keyword(s):  
Optical Properties ◽  
Structural Dynamics ◽  
Fluorescent Protein ◽  
Green Light ◽  
Endogenous Ligand ◽  
New Class ◽  
Fluorescence Protein

UnaG is a new class of fluorescence protein for which an endogenous ligand namely bilirubin (BLR) is playing the role of the chromophore. Upon photoexcitation, holoUnaG emits the green light....

scholarly journals Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis

2019 ◽  
Author(s):  
Yingshuo Hou ◽  
Siyu Chen ◽  
Jianjun Wang ◽  
Guizhen Liu ◽  
Sheng Wu ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Molecular Chaperone ◽  
Coenzyme A ◽  
Industrial Applications ◽  
Upstream Sequence ◽  
Activity Levels ◽  
Corynebacterium Ammoniagenes ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Dna Promoters

Abstract Background：Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by Corynebacterium ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved and the available constitutive promoters are rather limited in this strain. Results：In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels resulting in different fluorescent intensities. A fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.1 times. Conclusions：This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P &lt; 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

scholarly journals Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

2014 ◽  
Vol 60 (3) ◽  
pp. 230-237 ◽  
Author(s):  
Hitomi MATSUNARI ◽  
Toshihiro KOBAYASHI ◽  
Masahito WATANABE ◽  
Kazuhiro UMEYAMA ◽  
Kazuaki NAKANO ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transgenic Pigs ◽  
Specific Expression ◽  
Green Fluorescent

scholarly journals The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

2016 ◽  
Vol 7 ◽  
Author(s):  
Mariamichela Lanzilli ◽  
Giuliana Donadio ◽  
Roberta Addevico ◽  
Anella Saggese ◽  
Giuseppina Cangiano ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein

56 ESTABLISHMENT OF TRANSGENIC RED FLUORESCENCE PROTEIN (RFP) CLONE DOGS THROUGH A STABLE TRANSMISSION OF RFP GENE TO NEXT GENERATION

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
G. A. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Second Generation ◽  
Pcr Analysis ◽  
Next Generation ◽  
Wild Type ◽  
Red Fluorescence ◽  
Reproductive Ability ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Regulated Gene Expression ◽  
Stable Transmission

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.

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