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Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound.
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Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein(EGFP)as a marker.Methods The total RNA was extracted from Microsporum canis,and reversely transcribed into cDNA.The PQ-LRP gene was amplified by PCR using the above cDNA as the template.The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300.Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation,in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc.Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein.Results The expression vector pCAMBIA-LRP-EGFP was successfully constructed,and the fusion gene LRP-EGFP was expressed integratedly in Microsporum canis.Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis,giving a granular or cluster-like appearance.Conclusion The infusion gene LRP-EGFP can be successfully expressed in Microsporum canis,and PQ-LRP protein is located on the cell membrane of Microsporum canis.
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ABSTRACT Purpose: To identify the fetal stem cell (FSC) response to maternal renal injury with emphasis on renal integrity improvement and Y chromosome detection in damaged maternal kidney. Materials and Methods: Eight non-green fluorescent protein (GFP) transgenic Sprague-Dawley rats were mated with GFP-positive transgenic male rats. Renal damage was induced on the right kidney at gestational day 11. The same procedure was performed in eight non-pregnant rats as control group. Three months after delivery, right ne- phrectomy was performed in order to evaluate the injured kidney. The fresh perfused kidneys were stained with anti-GFP antibody. Polymerase chain reaction (PCR) assay was also performed for the Y chromosome detection. Cell culture was performed to detect the GFP-positive cells. Technetium-99m-DMSA renal scan and single-photon emission computed tomography (SPECT) were performed after renal damage induction and 3 months later to evaluate the improvement of renal integrity. Results: The presence of FSCs was confirmed by immune histochemical staining as well as immunofluorescent imaging of the damaged part. Gradient PCR of female rat purified DNA demonstrated the presence of Y-chromosome in the damaged maternal kidney. Moreover, the culture of kidney cells showed GPF- positive cells by immuno- fluorescence microscopy. The acute renal scar was repaired and the integrity of dam- aged kidney reached to near normal levels in experimental group as shown in DMSA scan. However, no significant improvement was observed in control group. Conclusion: FSC seems to be the main mechanism in repairing of the maternal renal injury during pregnancy as indicated by Y chromosome and GFP-positive cells in the sub-cultured medium.
Subject(s)
Animals , Male , Female , Pregnancy , Wound Healing/physiology , Chimerism , Fetal Stem Cells/physiology , Kidney Diseases/physiopathology , Maternal-Fetal Exchange/physiology , Time Factors , Y Chromosome , Immunohistochemistry , Tomography, Emission-Computed, Single-Photon , Cells, Cultured , Polymerase Chain Reaction , Fluorescent Antibody Technique , Rats, Sprague-Dawley , Radiopharmaceuticals , Technetium Tc 99m Dimercaptosuccinic Acid , Disease Models, Animal , Kidney Diseases/pathology , Kidney Diseases/diagnostic imagingABSTRACT
Objective To compare the regulation effects of different activated and inhibitory riboswitches , and to facilitate the precise regulation of gene circuits .Methods A green fluorescent protein amcyan expression vector regulated by different riboswitches (addA, M6, TPP and btuB) was constructed, and the expression level of amcyan under different ligand concentrations was analyzed by RT-qPCR and relative fluorescence intensity , and then compared with the expression level of a vector without any riboswitch .The dynamic control performance was analyzed .Results Under the control of addA and M6 activated riboswitches , the expression of green fluorescent protein increased with ligand concentrations , and addA riboswitch had more dynamic regulatory effect than M 6 riboswitch.However, under the control of TPP and btuB inhibitory riboswitches , the expression of green fluorescence decreased with the increase in ligand concentrations , and the dynamic regulation of btuB riboswitch was slightly greater than that of TPP riboswitch .Conclusion The regulation efficacy of different riboswitches which have the same mechanism varies .Activated riboswitch addA and inhibitory riboswitch btuB with dynamic regulation and control advantages are more suitable for precise metabolism regulation and target gene expression in Escherichia coli.
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Objective To explore a high-throughput method for quantitative analysis of autophagosomes.Methods Green fluorescent protein-light chain 3(GFP-LC3)transgenic murine keratinocytes were randomly divided into 4 groups:control group receiving no treatment,starvation group subjected to starved culture,20 J/cm2 ultraviolet A (UVA) group treated with 20 J/cm2 UVA radiation,and 40 J/cm2 UVA group treated with 40 J/cm2 UVA radiation.After 6-hour treatment,the cells were fixed,and images were acquired by confocal laser scanning microscopy.A macro was created by the ImageJ software to automatically quantify the GFP-LC3 puncta in the cells and the number of cells.Then,the level of autophagy was compared among different groups.Results By using the macro created by the ImageJ software,autophago-somes in the keratinocytes were successfully identified and quantified.Less than 0.6 second was needed for analyzing an image of 4.2 mega pixels in a test computer.The average number of autophagosomes in keratinocytes was significantly higher in the starvation group,20-J/cm2 UVA group and 40-J/cm2 UVA group than in the control group whether with the treatment with pepstatin A (F =20.05,P ＜0.05) or not (F =5.01,P ＜ 0.05).This method could successfully differentiate the autophagy levels among the starvation group,UVA irradiation groups and control group.Conclusion A new high-throughput method,which can rapidly and accurately quantify GFP-LC3 puncta in cells,is established successfully to quantificationally detect autophagy.
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Objective To detect the transferred vascular endothelial growth factor (VEGF)165 gene expression in rhesus autologous bone marrow mesenchymal stem cells (MSCs), and to explore the functional viability of transgenic MSCs. Methods MSCs from rhesus bone were isolated by Ficoll, which were used to detect the phenotype. After the culturing, the expression vector pcDNA-eGFP-VEGF165 was transfected into bone marrow MSCs. Fluorescence microscope and flow cytometry were used to detect the enhanced green fluorescent protein (eGFP) expression. At the same time, the phenotype in transfected MSCs was also indentified. The VEGF165 expression level was detected by RT-PCR. Results The highly purified MSCs were collected successfully. The transfected MSCs and daughter cells showed expressions of eGFP and VEGF165, which also remained the characteristics of MSCs. Conclusion The VEGF165 gene that is transfected into MSCs can maintain characteristics of MSCs, and stably express foreign genes.
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BACKGROUND:Previous studies have shown that a certain dose of acidic fibroblast growth factor can promote skeletal muscle satelite cel proliferationin vitro. OBJECTIVE:To investigate the effects of transfection with acidic fibroblast growth factor by electroporation on growth, proliferation and differentiation of skeletal muscle satelite cels. METHODS: Skeletal muscle satelite cels were cultured and purified, and then transfected with plasmid pSectag-GFP-aFGF by electroporation. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was calculated. After transfection, cel cycle was analyzed by flow cytometry to draw the growth curve of skeletal muscle satelite cels. Western blot assay was employed to measure protein level of acidic fibroblast growth factor. RESULTS AND CONCLUSION: (1) Immunocytochemistry detection: The skeletal muscle satelite cels were positive for a-sarcomeric actin. (2) Transfection efficiency: At 12 hours after transfection with pSectag-aFGF, several cels showed green fluorescence, and the green fluorescent expression reached the peak at 72-96 hours after transfection with a positive rate of about 90%. (3) Cel cycle: After electrotransfection, the proportion of cels at S phase in the electroporation group was higher than that in the control group (P < 0.05). (4) Cel growth curve: At 3 days after electrotransfection, the cels entered logarithmic growth phase but the proliferation slowed down at 5 days. (5) Differentiation capacity: There were fewer myotubes and aging cels in the electroporation group than the control group. (6) Western blot assay: Acidic fibroblast growth factor protein was highly expressed in the cels transfected with target gene detected by western blot assay. These findings indicate that by using electroporation method, acidic fibroblast growth factor can be transferred into skeletal muscle satelite cels and have a high-efficiency and long-term expression, which can promote the proliferation of skeletal muscle satelite cels and inhibit formation of myotubes.
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Objective To construct a lentiviral expression vector of peroxiredoxin2(PRDX2) RNA interference (RNAi) and to investigate the effect of siRNA of PRDX2 genes on the proliferation of human colonrectal cancer SW480 cell .Methods RNAi tar‐get sequences were designed and synthesized towards the PRDX2 gene sequences .The lentiviral vector pGC‐EGFP‐shPRDX2 was constructed and identified .The vector was transformed into SW480 cells ,and the transfection efficiency was evaluated by fluores‐cence microscopy .The expression of PRDX2 was detected with Quantitative real‐time PCR (qRT‐PCR) and Western blot in the transfected cells .Cell growth and colony forming ability were detected with MTT and plate cloning technique .Results PRDX2 gene lentiviral vector was successfully established and was proved by gene sequencing .The expression of PRDX2 in mRNA and pro‐tein was significantly reduced(P<0 .05) .The PRDX2 mRNA and protein expression in SW480 transfected with lentiviral were sig‐nificantly reduced (P< 0 .05) ,and the ability of growth and proliferation were significantly reduced(P< 0 .05) .Conclusion PRDX2 gene lentiviral vector could be a stable and reliable tool .The proliferation and growth of SW480 cells transfected by pGC‐EGFP‐shPRDX2 could be effectively suppressed ,which could facilitate further investigation of the roles of PRDX2 gene in the de‐velopment and progression of colorectal cancer .
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BACKGROUND:Kalikrein 1 is an important component of the kalikrein-kinin system. Studies have shown that kalikrein can protect the cardiovascular system by promoting angiogenesis and inhibiting myocardial inflammation, but there is no report on its effect on inducing differentiation of stem cels. OBJECTIVE: To determine the transfection efficiency of kalikrein 1 adenoviral vector in rat bone mesenchymal stem cels. METHODS:Using adenovirus as a vector, the target gene kalikrein 1 was transfected into rat bone marrow mesenchymal stem cels. Fluorescence microscopy, MTT method and flow cytometry were used to investigate the effect of transfection and determine the optimal multiplicity of infection. RESULTS AND CONCLUSION:Adenovirus carrying kalikrein 1 was successfuly transfected into rat bone marrow mesenchymal stem cels. Results from flow cytometry showed that the transfection efficiency was associated with the multiplicity of infection. When the multiplicity of infection was 150, the transfection efficiency was 80.8%. MTT results showed that when the multiplicity of infection was 200, the cel growth was inhibited remarkably. These findings indicate that adenovirus-mediated kalikrein 1 can be successfuly transfected into rat bone marrow mesenchymal stem cels with the optimal multiplicity of infection=150.
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Objective To compare different ways to trace bone marrow mesenchymal stem cells (BMSCs) after being transplanted in cerebral ischemia-reperfusion injury. Methods Male SD rats of SPF grade were randomly divided into sham group, model group (ischemia-reperfusion,IR), BrdU tracing group, PKH26 tracing group and GFP tracing group. Fo?cal cerebral ischemia-reperfusion model was established by blocking middle cerebral artery. 24 hours after cerebral isch?emia-reperfusion injury, 10μL BMSCs that were labeled respectively by BrdU, PKH26, GFP were added respectively into BrdU, PKH26 and GFP tracing group while equal volum of normal saline was added into sham group and model group. Mod?el and transplanting cells efficacy was determined by neural behavioral score, TTC staining and brain water content;Neurons were counted using tar violet staining;The number of transplant cells in the transplanting site was assessed by fluorescence microscopy. Results Before transplanting, there was no significant difference among BrdU, PKH26 and GFP group in cell labeled efficacy. By contrast, neural behavioral score, brain infarct volume and brain tissue water content were significantly lower in all three tracing groups than that in model group 4 weeks after transplantation while neuron counts were markedly higher. There was no significant difference of above parameters among the three tracing groups. However, the number of traced transplanting cells in damaging area in GFP group is significantly higher than that in BrdU group and PKH26 group. Conclusion In cerebral ischemia-reperfusion injury, the tracing effect of GFP last longer, therefore it is significantly more effective than BrdU and PKH26.
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BACKGROUND:Previous studies have suggested that achaete-scute homology 1 (ASCL1) plays a key role in the neuronal commitment. Therefore, somatic cels may directly differentiate into neurons by gene transfection ofASCL1, which wil provide new therapeutic strategies for optic nerve regeneration. OBJECTIVE:To construct a recombinant adenovirus vector expressing ratASCL1 gene for further research ofASCL1 gene function. METHODS:The ratASCL1 gene and advenovirus shuttle plasmid (pYr-adshuttle-4) which contained enhanced green fluorescent protein (EGFP) reporter gene were cleaved by restriction endonucleaseXhoI andEcoR I. The target gene fragments were connected together to generate a recombinant plasmid pYr-ads-4-rat-ASCL1 and then transfected into E.coliDH5α. The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequence reaction. The plasmid pYr-ads-4-rat-ASCL1 and pAd/PL-DEST were reconstructed by homologous recombination processes to obtain rat ASCL1 recombinant adenovirus vector. The plasmid pYrAd-rASCL1 was linearized byPac I and subsequently transfected into HEK293 cels for packaging and amplification. RatASCL1 gene in the recombinant adenoviruses were identified by PCR. Virus titer was determined by tissue culture infectious dose 50. Infection efficiency was monitored by EGFP expression. RESULTS AND CONCLUSION:Restriction endonuclease digestion and DNA sequencing showed that the recombinant adenovirus vector pAd-rat-ASCL1 was constructed correctly. The positive amplification bands of 862 bp could be seen in PCR analysis. The virus titer reached 2×1010 pfu/mL. Infection efficiency of recombinant adenovirus in HEK293 cels was more than 80%. The results indicate that the recombinant the adenovirus vector containingASCL1 with high titer and infection efficiency has been successfuly constructed, which can be helpful for further research of the function and clinical application ofASCL1 gene for optic nerve regeneration.
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Objective To observe the effects of green fluorescence protein mediated by lentivirus in bladder, and to de?termine the amount of virus obtained good transfection effects. Methods lentivirus carring GFP gene was perfused using transurethral approach into bladder of guinea pigs. Samples of bladder, liver, kidney and lungs were collected for frozen sec?tions after feeding seven days. The distribution of green fluorescence was observed using laser confocal microscopy. Re?sults The titer of lentivirus was 4 × 108. GFP was found under the mucosa when the amount of lentivirus transurethral was 30μL. GFP was distributed widely in muscle layer using 40μL lentivirus. GFP was detected even stronger in muscle layer when the amount was 50μL. GFP was found in muscle layer when 25μL lentivirus was injected intravenously. GFP was not found in other tissues than in bladder via transurethral perfusion. There was higher GFP in liver, lungs and other organs than in bladder via intravenous injection. Conclusion Lentivirus can mediate GFP transfecting bladder of guinea pig successful?ly and escape the distribution of GFP all over the body intravenously, which will bring new research direction and method for clinical treatment of diseases in bladder.
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Objective:To explore whether cigarette smoking extract (CSE) has influence on fluorescence protein ex-pression of thrombomodulin (TM) on surface of African green monkey kidney fibroblast cells (COS-7) or not . Methods:When TM-green fluorescent protein (GFP) plasmid was successfully constructed ,COS-7 cells were trans-fected by it ,then incubated by prepared 5% CSE (5% CSE group) ,PBS of certain volume was added to serum-free minimal essential medium (MEM ,simple medium) ,which was cultured at the same time and treated as control group .Flow cytometry counting method was used to detect change of TM-GFP expression amount on COS-7 surface at different time point .Results:Compare with control group ,there were no significant difference in the expression of TM-GFP on COS-7 surface at 1h and 6h in 5% CSE group [1h :(134.99 ± 18.41) vs .(146.61 ± 12.06) ,6h :(116.89 ± 27.28) vs .(123.89 ± 39.24) ,P>0.05 both] .Conclusion:The 5% cigarette smoking extract has no influ-ence on fluorescence expression of thrombomodulin on surface of African green monkey kidney fibroblast cells .
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BACKGROUND:Studies have shown that human leukocyte antigen (HLA)-A*0206 subtype is related to the abscess of nasopharyngeal carcinoma, but there is no corresponding transgenic animal models that could used to judge the relationship between HLA-A*0206 and nasopharyngeal carcinoma on the overal level and further research of immunotherapy and gene therapy. OBJECTIVE:To construct lentiviral vectors carrying pLVX-CMV-HLA-A*0206-HA-mCMV-ZsGreen and establish HLA-A*0206 transgenic mice. METHODS:The HLA-A*0206 sequence was synthesized. EcoRI recognition site was introduced in the 5’ end by polymerase chain reaction, and influenza virus hemagglutinin labels and BamHI recognition site were introduced in the 3’ end. Eco RI and Bam HI double enzyme digestion target fragments and the pLVX-CMV-mCMV-ZsGreen plasmids were connected to the digested productions and transfected JM109 competent cel s. The positive clones were selected and identified by double enzyme digestion and sequencing. The positive plasmid and packaging plasmids were transfected into 293T cel s, which were human renal epithelial cel line that can express SV40 large T antigen. The lentivirus containing target sequence was produced. RESULTS AND CONCLUSION:Gel electrophoresis and sequencing results showed that, HLA-A*0206 was successful y inserted into pLVX-CMV-mCMV-ZsGreen frame plasmids. Transfection efficiency was 92%after 48 hours of transfecting 293T cel s. The viral suspension titer was 5 × 108 measured by fluorescence method. Experimental findings indicate that, the lentivirus containing cytomegalovirus promoter, HLA-A*0206, influenza virus hemagglutinin label and Zsgreen report gene was successful y constructed.
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BACKGROUND:It is vital to choose the appropriate carrier with low toxicity and high gene transfection efficiency in gene therapy, which is harmless to human body and environment. OBJECTIVE: To prepare superparamagnetic Fe3O4/SiO2-polyethyleneimine (PEI) composite particles. METHODS: Fe3O4 nanoparticles were prepared via an emulsion solvent evaporation method and superparamagnetic Fe3O4/SiO2 core shel microspheres were prepared successfuly subsequently via a modified stober method. The microspheres were further modified with PEI to obtain superparamagnetic Fe3O4/SiO2-PEI composite particles. The structures and properties of resultant composite particles microspheres were characterized by transmission electron microscopy, zeta potential and vibrating sample magnetometer. Superparamagnetic Fe3O4/SiO2-PEI composite particles were mixed with plasmid DNA at different mass ratios (29∶1, 39∶1, 49∶1, 59∶1, 68∶1, 78∶1, 88∶1). Thein vitro gene transfection ability was evaluated by Hela cels with the transfection of plasmid DNA encoded with green fluorescent protein and the transfection efficiency was determined by confocal fluorescence microscopy. RESULTS AND CONCLUSION: We successfuly synthesized the Fe3O4/SiO2-PEI composite particles with good dispersibility and even size distribution (about 100 nm). The surface charge was 21.07 mV, and the saturation magnetization was 28.05 emu/g that meant superparamagnetism. When the mass ratio was 59∶1, al the plasmid DNA was adherent to the Fe3O4/SiO2-PEI composite particles; when the mass ratio was > 59∶1, there were excessive Fe3O4/SiO2-PEI composite particles. Therefore, the mass ratio of 59:1 could lead to a better outcome for HeLa celltransfection. These results indicate that the Fe3O4/SiO2-PEI composite particles can dramaticaly improve the transfection efficiency of plasmid DNA compared with PEI.
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BACKGROUND:Human umbilical vein endothelial cells transfected with lentivirus containing enhanced green fluorescent protein can be easily traced. The optimal multiplicity of infection and time for producing strong fluorescence intensity can lay the foundation of tracing human umbilical vein endothelial cells in animal models. OBJECTIVE:To observe expression of lentivirus containing enhanced green fluorescent protein in human umbilical vein endothelial cells, and thereby to find a stable method to label human umbilical vein endothelial cells. METHODS:Using 0.1%col agenase perfusion digestion, we isolated human umbilical vein endothelial cells, which then were placed into a culture medium containing 20%fetal bovine serum and endothelial cellgrowth factor and observed under an inverted microscope. Fol owing digestion, centrifugation and suspension, the cells were counted and divided into four groups, 5.0×105 cells in each group. After cells were seededonto 24-wel plates, 10μL serum-free Dulbecco’s modified Eagle’s medium was added into the blank group, and lentiviruses containing enhanced green fluorescent protein were added into another three groups for celltransfection respectively at multiplicities of infection of 2, 3, 4. There were three dishes in each group. RESULTS AND CONCLUSION:After cultured for 5-7 days, isolated cells grew into a single layer and exhibited a cobblestone-like arrangement under a light microscope. In addition, factor VIII related antigen test was positive. A green fluorescence was visible at 24 hours of transfection, and peaked at 72 hours. Transfection efficiency was in a linear growth with the multiplicity of infection. Up to the 21st day of transfection, the green fluorescence was stil visible. After 0, 7, 14, 21 days of transfection, the number of human umbilical vein endothelial cells showed no difference between the transfection group with the multiplicity of infection=3 and blank group, suggesting the proliferative ability of cells has no changes after transfection with lentivirus containing enhanced green fluorescent protein. These findings indicate that the lentivirus containing enhanced green fluorescent protein can highly transfect human umbilical vein endothelial cells, and green fluorescent protein can sustainably express for 21 days but cannot impact the cellproliferation.
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Objective To investigate the biological features of the mouse bone marrow stromal stem cells (BMSCs) transfected by humanβnerve growth factor(β-NGF) .Methods BMSCs of GFP transgenic mouse were isolated and cultured .Theβ-NGF re-combinant plasmid vectors were transferred into the cultured BMSCs by LipofectamineTM 2000 .The expression of β-NGF was detec-ted with ELISA .Results The β-NGF recombinant plasmid vectors were successfully transferred into BMSCs of GFP transgenic mouse ,the expression of β-NGF in transfected cells appeared .In addition ,the expression ofβ-NGF could effectively protect the BM-SCs which were injured in transfection process .Conclusion BMSCs of GFP transgenic mouse after transfecton with human β-NGF have the proliferation and differentiation capacity ,and possess the expression ability of β-NGF protein .
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Objective To investigate the effectiveness and toxicity of the transfection of lentiviral vector-mediated enhanced green fluorescent protein (LV-EGFP) to rat cornea with different concentrations and methods, and to look for the most effective way for transfecting the virus to rat cornea. Methods Twenty-five SD rats were randomly divided into five groups, namely MOI=5 eyedrop group (group A), MOI=5 subconjunctival injection group (group B), MOI=10 eyedrop group (group C), MOI=10 subconjunctival injection group (group D), and MOI=10 in vitro transfection group (group E). Rats in each group were treated with corresponding concentration of LV-EGFP respectively. The distribution and intensity of fluorescence in the corneas were observed under inverted fluorescence microscope, and the corneal cellular morphology was observed with HE staining 7 days after transfection. Results Within the same MOI groups, fluorescence spread more uniformly in eyedrop groups as compared with that in subconjunctival injection groups. The fluorescence intensity (A value) in five groups were 0.1803±0.0440, 0.1061±0.0434, 0.2369±0.0157, 0.2002±0.0307, 0.2434±0.0173 respectively, which was stronger in eyedrop groups than in subconjunctival injection groups (P<0.05). In high MOI groups more EGFP could be expressed as compared with low MOI groups (P<0.05). The fluorescence intensity was significantly higher in group E than in groups A, B and D, while there was no statistical significant difference between groups E and C. HE staining showed no apoptotic corneal endothelial cells. The corneal endothelial cells were still growing well with no shrinkage, deformation and loss in vitro 7 days after transfection. Conclusion Rat cornea could be effectively transfected by LV-EGFP with low MOI. Eyedrop method is found to have a higher efficiency compared with subconjunctival injection. An increase in amount of MOI can increase transfection efficiency. Continuous transfection with low MOI to cornea of rat is safe.
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Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.
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BACKGROUND:Mesenchymal stem cells from transgenic animals carry green fluorescent protein that can be stably expressed in viable cells and can be used to rapidly screen modified cells in comparison with traditional virus and plasmids. OBJECTIVE:To study the biological features of bone marrow derived stromal cells from transgenic rats with green fluorescent protein gene. METHODS:Bone marrows were isolated from the bilateral long bones of 2-week-old transgenic rats with green fluorescent protein gene, and cultivated in conditioned culture medium to obtain bone marrow mesenchymal stem cells with green fluorescent protein gene. The passage 5 bone marrow mesenchymal stem cells with green fluorescent protein gene were analyzed by flow cytometry to detect cellsurface molecules, and were induced in calcium or adipogenic induction medium. After calcium induction, the alizarin red staining was performed to test the formation of calcium concentration. After adipogenic induction, bone marrow mesenchymal stem cells were detected with oil red O staining. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells, stably expressing green fluorescent protein gene, were obtained successful y from the transgenic rat with green fluorescent protein gene. The cells expressed CD90, CD105 strongly, but did not express or weakly expressed CD14 andCD45. After 3 weeks of calcium induction, jacinth calcium salts could be detected in the cells by alizarin red staining. At 3 weeks of adipogenic induction, the cells showed positive oil red O staining. The green fluorescent protein gene, as report gene, exerts no effect on the cellsurface molecules and multilineage potential of bone marrow mesenchymal stem cells, which can be used as an efficient tracer.