423 PRODUCTION OF CLONED PIGS EXPRESSING APOLIPOPROTEIN E-SPECIFIC SMALL HAIRPIN (shRNA)

2010 ◽  
Vol 22 (1) ◽  
pp. 369 ◽  
Author(s):  
M. El-Beirouthi ◽  
M. S. Albornoz ◽  
M. A. Martinez-Diaz ◽  
D. Zadworny ◽  
L. B. Agellon ◽  
...  
Keyword(s):  
Apolipoprotein E ◽  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Fluorescent Light ◽  
Gfp Expression ◽  
Apo E ◽  
The Status ◽  
Morphological Defects ◽  
Green Fluorescent

Apolipoprotein E (apo E) is a known risk factor for developing premature atherosclerosis and Alzheimer’s syndrome. The aim of this study was to create a pig model with reduced apo E levels using RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies. Three synthetic small interfering RNA targeting the porcine apo E mRNA were designed, and the knockdown efficiency was assessed in cultured porcine granulosa cells by real-time PCR. The observed apo E knockdown efficiency ranged from 45 to 82% compared with control cells, indicating the targeted degradation of apoE mRNA.A small hairpin RNA (shRNA) expressing vector was constructed in PRNAT.U6.Neo (Genscript Corp., Piscataway, NJ, USA) based on the most effective apo E RNAi sequence under the control of polymerase III (U6) promoter, and then introduced into fetal porcine fibroblast cells. Clones were selected by neomycin treatment and green fluorescent protein (GFP) expression. SCNT was performed using IVM oocytes collected from prepubertal gilts. Oocyte maturation and activation and embryo culture were performed as previously described (Nascimento et al. 2009 Reprod. Domest. Anim. in press). Embryos were cultured in vitro for 5 to 6 days, briefly exposed to fluorescent light to confirm GFP expression, and then surgically transferred into the uterus of recipient gilts. The recipient gilts were synchronized by daily oral administration of altrenogest (20 mg day-1; Regu-Mate®, Intervet, Millsboro, MD, USA) for 12 or 13 days, followed by 1000 IU of eCG injected in the last day of altrenogest treatment and 500 IU of hCG 72 h later. Pregnancy diagnosis was performed by ultrasonography at Day 20 to 25 after embryo transfer, and parturition was induced by injecting PGF2? (10 mg of dinoprost tromethamine; Lutalyse®, Pfizer Canada Inc., Montreal, QC, Canada) at Day 115 of pregnancy. Rates of cleavage (74.7%) and development to the blastocyst stage (37.2%) were comparable with that of embryos reconstructed with nontransfected cells from the same cell line. A total of 309 embryos were transferred to 5 recipients, of which 3 became pregnant and farrowed. Seven live and 1 stillborn piglets were delivered naturally. The presence of the introduced plasmid and the expression of the GFP transgene tag were confirmed by PCR in placental and umbilical tissues of all the piglets. Six cloned pigs have survived after weaning and exhibit no obvious morphological defects. The status of apo E gene expression is currently under investigation. Supported by a NSERC Discovery Grant to VB.

scholarly journals Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Shahin Eghbalsaied ◽  
Kamran Ghaedi ◽  
Götz Laible ◽  
Sayed Morteza Hosseini ◽  
Mohsen Forouzanfar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Gfp Expression ◽  
Exogenous Dna ◽  
Bovine Sperm ◽  
Gfp Fluorescence ◽  
Gfp Reporter ◽  
Green Fluorescent

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

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

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.

scholarly journals In vivo analysis of key elements within the renin regulatory region

2008 ◽  
Vol 35 (3) ◽  
pp. 243-253 ◽  
Author(s):  
Sean T. Glenn ◽  
Craig A. Jones ◽  
Li Pan ◽  
Kenneth W. Gross
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Region ◽  
Renin Angiotensin System ◽  
Artificial Chromosome ◽  
Proximal Promoter ◽  
Gfp Expression ◽  
Renin Gene ◽  
Green Fluorescent

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

scholarly journals Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820 ◽  
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cns Infection ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Differential Gene ◽  
Green Fluorescent

ABSTRACT Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

In vivo and in vitro efficacy of capecitabine (X) + tamoxifen (TAM) in breast cancer (BC)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21092-21092
Author(s):  
K. Mori ◽  
Y. Yamaguchi ◽  
N. Sawada ◽  
K. Kondoh ◽  
S. Hayashi
Keyword(s):  
Breast Cancer ◽  
Fluorescent Protein ◽  
Active Form ◽  
Signal System ◽  
Gfp Expression ◽  
Responsive Element ◽  
Green Fluorescent ◽  
Mcf 7

21092 Background: In vitro studies in BC cell lines suggested antagonism between TAM and 5-FU. Thymidine phosphorylase (TP) activates X to 5-FU in tumors. X activity correlates with tumor TP concentrations in vivo. Methods: We studied antitumor efficacy of X + TAM in vivo and in vitro in human BC models. Nude mice were inoculated s.c. with estradiol, then MCF-7 cells 1 day later. When tumors were ∼300 mm3, mice received 6 weeks’ oral vehicle (control), X (d1–14 q21d) at MTD (539 mg/kg) or 2/3 MTD, and/or TAM at 100 or 30 mg/kg/d. We also analyzed impact of 5-FU and doxifluridine (5'-DFUR, an intermediate of X) + TAM on estrogen receptor (ER) signals in an in-vitro culture system. ER signals were monitored by expression of green fluorescent protein (GFP) in MCF-7 BC cells transfected with the estrogen-responsive element (ERE)-GFP gene (MCF-7-E10). GFP expression was induced in MCF-7-E10 cells in the presence of estradiol at 3 pM or BC tissue supernatant. Results: X at 2/3 MTD + TAM 30 mg/kg were significantly more active than the highest dose of X or TAM alone. Tumor TP concentrations were significantly higher in TAM- than vehicle-treated mice. In the ER signal system, GFP expression of MCF-7-E10 was reduced by 4-hydroxytamoxifen (4-OHT, active form of TAM) at 0.01 and 0.1 nM. When added to 4-OHT, 5-FU 0.3–30 μM or 5'-DFUR 3–10 μM reduced GFP expression more than either agent alone. In vitro, 5-FU and 5'-DFUR inhibited proliferation of MCF-7-E10 cells regardless of 4-OHT. Additive effects could not be confirmed as 4-OHT alone showed only marginal anti- proliferative activity at 0.01–0.1 nM. Conclusion: X and TAM are not antagonistic in this model. TAM may augment X activity via TP upregulation in BC tissues. TAM and X intermediates showed no clear antagonism in vitro in an ER signal system. All-oral X + TAM merits evaluation as combination therapy in breast cancer. [Table: see text]

scholarly journals 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

2000 ◽  
Vol 66 (9) ◽  
pp. 4074-4083 ◽  
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.

307 TRANSGENIC OVINE EMBRYOS BY ARTIFICIAL INSEMINATION, IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION

2009 ◽  
Vol 21 (1) ◽  
pp. 250 ◽  
Author(s):  
F. Pereyra-Bonnet ◽  
A. Gibbons ◽  
M. Cueto ◽  
R. Fernández-Martín ◽  
D. Salamone
Keyword(s):  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Green Fluorescent Protein Gene ◽  
Sperm Cells ◽  
Normal Female ◽  
Embryo Viability ◽  
Vitro Fertilization ◽  
Transgenic Embryos ◽  
Green Fluorescent

Nowadays, transgenesis in animals constitutes an important tool for pharmacological protein production and livestock improvement. In 1971 Brackett first described that heterologous DNA can be introduced into a mammalian oocyte using sperm cells as vectors. We evaluated the capacity of AI, IVF and ICSI to produce transgenic embryos, in ovine, using sperm that had been exposed to a pCX-EGFP plasmid in Long and Short incubation protocols. The pCX-EGFP plasmid contains an enhanced green fluorescent protein gene (egfp) under the chimeric cytomegalovirus-IE-chicken β-actin enhancer-promoter control. Sperm/pCX-EGFP incubation was carried out by Long Incubation (2 h at 17°C in 200 μL of SFM medium: 100 mL contains glucose 1.2 g, Na citrate 1.0 g, EDTA 0.4 g, Citric acid 0.3 g, Trizma 0.6 g) and Short Incubation (5 min at 0–5°C in 10–100 μL of extender medium: 100 mL contains Na Citrate 2.8 g and EDTA 4 mg). For AI, Merino sheep (n = 17) were superovulated and inseminated with fresh semen (200 millions sperm/sheep) from eight Merino rams. The embryos were recovered by flushing the uterine horns by standard procedures. In IVF and ICSI, slaughterhouse oocytes were fertilized with frozen/thawed sperm. IVF was carried out in Brackett-Oliphant medium with 5 mm of caffeine, 20 IU mL–1 of heparin with 20 million sperm mL–1 during 5 h in an atmosphere of 5% CO2 in air. In ICSI, the spermatozoon was immobilized by breaking its tail and injected into MII oocytes. Immediately the oocytes were activated by incubation in TALP-HEPES with 5 μm ionomycin for 4 min, cultured in TCM199 for 3 h and transferred to a droplet of 1.9 mm DMAP for 3 h. Maturation and cultivation conditions were determined by standard operating procedures. All embryos were exposed to blue light (488 nm) to determine the percentage of morulae/blastocysts showing green fluorescence. Results are shown in Table 1. Statistical analysis was done by Fisher test. AI and IVF were able to produce a high percentage of morula and blastocyst stage, but were unable to produce transgenic embryos. In contrast, regardless of the sperm/plasmid incubation protocol, high percentages of transgenic morulae and blastocysts were always obtained by ICSI and the highest rate was achieved with Short Incubation (P < 0.05). In order to demonstrate ICSI-Short incubation embryo viability, two-day-old non-selected fluorescence embryos (n = 45), were transferred into the oviducts of five surrogate mothers. Pregnancy was diagnosed at day 25 (2/5; 40%), and one normal female lamb was recently born (1/5; 20%). In conclusion, our results show that in ovine, ICSI seems to be the only method for producing transgenic embryos using sperm cells as vectors. In addition the offspring born confirm the viability of these embryos. Table 1.Development and fluorescence expression of ovine embryos

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 339-346 ◽  
Author(s):  
Liangxue Lai ◽  
Qingyuan Sun ◽  
Guangming Wu ◽  
Clifton N. Murphy ◽  
Birgit Kühholzer ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Pronuclear Formation

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8±17.3% vs 28.5±3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0±7.0% vs 63.3±12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0±11.6% vs 4.6±4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Disulphide-less crotamine is effective for formation of DNA–peptide complex but is unable to improve bovine embryo transfection

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 72-79
Author(s):  
Vicente J.F. Freitas ◽  
Iana S. Campelo ◽  
Mirelly M.A.S. Silva ◽  
Camila M. Cavalcanti ◽  
Dárcio I.A. Teixeira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Peptide Sequence ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Gfp Expression ◽  
Green Fluorescent ◽  
Exogenous Gene

SummaryThis study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

scholarly journals Analysis of Green Fluorescent Protein Expression in Transgenic Rats for Tracking Transplanted Neural Stem/Progenitor Cells

2005 ◽  
Vol 53 (10) ◽  
pp. 1215-1226 ◽  
Author(s):  
Andrea J. Mothe ◽  
Iris Kulbatski ◽  
Rita L. van Bendegem ◽  
Linda Lee ◽  
Eiji Kobayashi ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Green Fluorescent Protein ◽  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Rat Strain ◽  
Direct Fluorescence ◽  
Green Fluorescent

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents—Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]—by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wildtype spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

Sign in / Sign up

Export Citation Format

Share Document