Green fluorescent protein as a reporter for gene transfer studies in the cochlea

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

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Gene Transfer in Rodent Nervous Tissue Following Hindlimb Intramuscular Delivery of Recombinant Adeno-Associated Virus Serotypes AAV2/6, AAV2/8, and AAV2/9

Neuroscience Insights ◽

10.1177/1179069519889022 ◽

2019 ◽

Vol 14 ◽

pp. 117906951988902 ◽

Cited By ~ 1

Author(s):

Asad Jan ◽

Mette Richner ◽

Christian B Vægter ◽

Jens R Nyengaard ◽

Poul H Jensen

Keyword(s):

Nervous System ◽

Viral Load ◽

Green Fluorescent Protein ◽

Gene Transfer ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Adeno Associated Virus ◽

Green Fluorescent ◽

Virus Delivery ◽

Intramuscular Delivery

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus–mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

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Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo

Blood ◽

10.1182/blood.v90.5.1777 ◽

1997 ◽

Vol 90 (5) ◽

pp. 1777-1786 ◽

Cited By ~ 167

Author(s):

Derek A. Persons ◽

James A. Allay ◽

Esther R. Allay ◽

Richard J. Smeyne ◽

Richard A. Ashmun ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

Fluorescent Protein ◽

High Titer ◽

Hematopoietic Cells ◽

Green Fluorescent Protein Gene ◽

Retroviral Gene Transfer ◽

Green Fluorescent ◽

Marrow Cells

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

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Novel Method of Gene Transfer in Birds: Intracytoplasmic Sperm Injection for Green Fluorescent Protein Expression in Quail Blastoderms1

Biology of Reproduction ◽

10.1095/biolreprod.110.085860 ◽

2010 ◽

Vol 83 (6) ◽

pp. 965-969 ◽

Cited By ~ 10

Author(s):

Shusei Mizushima ◽

Soichi Takagi ◽

Tamao Ono ◽

Yusuke Atsumi ◽

Akira Tsukada ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

Protein Expression ◽

Intracytoplasmic Sperm Injection ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Novel Method ◽

Green Fluorescent

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Green Fluorescent Protein as a Selectable Marker of Fibronectin-Facilitated Retroviral Gene Transfer in Primary Human T Lymphocytes

Human Gene Therapy ◽

10.1089/10430349950019147 ◽

1999 ◽

Vol 10 (1) ◽

pp. 5-14 ◽

Cited By ~ 59

Author(s):

Valerie Dardalhon ◽

Nelly Noraz ◽

Myriam Boyer ◽

Arjen Q. Bakker ◽

Karen Pollok ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

T Lymphocytes ◽

Fluorescent Protein ◽

Selectable Marker ◽

Retroviral Gene Transfer ◽

Human T Lymphocytes ◽

Green Fluorescent

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Transfer of Green Fluorescent Protein (GFP) Gene to Betta splendens Embryos by Transfection and Electroporation Methods

Omni-Akuatika ◽

10.20884/1.oa.2018.14.2.556 ◽

2018 ◽

Vol 14 (2) ◽

Author(s):

Eni Kusrini ◽

Alimuddin Alimuddin ◽

Erma Primanita Hayuningtyas ◽

Syuhada Restu Danupratama

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

Expression Vector ◽

Fluorescent Protein ◽

Betta Splendens ◽

Foreign Gene ◽

Gfp Expression ◽

Dna Concentration ◽

Gfp Gene ◽

Green Fluorescent

Transfection and electroporation method shave a high possibility to apply towards transgenic production of small eggs size fish species. This study aimed to examine the potential of transfection and electroporation methods to use for transferring a foreign gene into betta fish (Betta splendens) embryos using green fluorescent protein (GFP) gene as a model. Fish were spawned naturally in the ratio of male: female was 1:1, then a total of 200 eggs were taken for each treatment. Transfection was performed for 30 minutes (room temperature of about 25 °C) at two-cell stage of embryos using transfast reagent. Transfection reaction consisted of 0.75 µL transfast reagent, 0.25 µL GFP expression vector (DNA concentration: 50 µg/µL) and 99 µL NaCl solution (concentration: 0,95%). Electroporation was performed using 125 volt cm-1, 3 times pulse frequency at one second interval and pulse length of 7 micro seconds. A volume of 800 µL GFP expression vector solution (DNA concentration: 50 µg/ µL) in PBS was used for electroporation. The successful of foreign gene transfer was determined by PCR method with GFP specific primers. The results showed that hatching rate of eggs in transfection treatment was 67.08%, while the electroporation was 72.09%. Survival of larvae in transfection treatment was 73.00%, while the electroporation was 75.00%. The results of PCR analysis showed that transfection method allowed 65% of the survived fish carrying GFP gene, whereas the electroporation method was 70%. Thus, foreign gene transfer in betta fish can be conducted using the transfection and electroporation methods.

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Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614045 ◽

2000 ◽

Vol 84 (09) ◽

pp. 460-467 ◽

Cited By ~ 8

Author(s):

M. L. M. Lamfers ◽

M. J. Wijnberg ◽

J. M. Grimbergen ◽

L. G. M. Huisman ◽

M. C. Aalders ◽

...

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Green Fluorescent Protein ◽

Gene Transfer ◽

Receptor Binding ◽

Fluorescent Protein ◽

Transfection Efficiency ◽

Adenoviral Gene Transfer ◽

Amino Terminal ◽

Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

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161 EFFECTS OF LIPOSOMES ON SPERM MOTILITY AND DNA-BINDING EFFICIENCY

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab161 ◽

2017 ◽

Vol 29 (1) ◽

pp. 189

Author(s):

S. N. Lotti ◽

M. Rubessa ◽

R. V. Knox ◽

M. B. Wheeler

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

Dna Binding ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Selection Method ◽

Computer Assisted ◽

Exogenous Dna ◽

Liposome Preparation ◽

Green Fluorescent

In mice, microinjection is the most common gene transfer method used. Unfortunately, this strategy does not translate as well to livestock. Another potential method is sperm-mediated gene transfer, which takes advantage of sperm’s natural ability to bind to naked DNA. Gene transfer using sperm-mediated gene transfer has been shown in pigs (Gandolfi et al. 1989 J. Reprod. Fert. Abstr. Ser. 4) and cattle (Perez et al. 1991 Biotecnol. Apl. 8, 90–94). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analysed methods to select thawed bovine sperm for DNA binding and evaluated the binding of exogenous DNA to those sperm. To determine the optimal sperm-selection method, the sperm were analysed using a computer-assisted sperm analyzer (CASA), the parameters selected were: total motility, rapid motility, and progressive motility. To measure the binding of DNA we used an indirect analysis using NanoDrop technology (Thermo Scientific, Wilmington, DE, USA) to compare the different DNA concentrations among groups. Liposome preparation was done using a cationic lipid, 3-(trimethyl ammonium iodide) 1,2 dimystryl-propanediate and a neutral lipid, l-a Dioleoyl phosphatidyl-ethanolamine prepared according to the protocol of Russell (1997). Percoll or swim-up methods were used to select sperm after thawing (Rubessa et al. 2016), followed by incubation (3 h) with the liposome-DNA complexes according to liposome preparation protocol (Russell, 1997). We used enhanced green fluorescent protein in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analysed: 1) immediately after processing (Control), 2) After 3 h of incubation with no liposomes, 3) incubation with liposomes and no DNA, 4) incubation with 1 ng of DNA, and 5) incubation with 10 ng of DNA. This was repeated five times. The CASA results for total motility and rapid motility showed a greater amount of significant differences (P < 0.01) between the control and the other treatments in the Percol group as opposed to swim-up. These results confirm that the sperm selected with swim-up is more stable. Following CASA analysis, sperm was washed with PBS twice and collected in tubes. The DNA from all samples was extracted to determine the quantity of attaching varying amounts of DNA to sperm. The results showed a general increase in DNA concentrations with the increase of DNA added for both methods, but the statistical variation was too large to draw any definite conclusion. In future studies, real-time PCR will be used to determine the quantity of enhanced green fluorescent protein bound to the sperm. Table 1. Results of the computer-assisted sperm analyzer (CASA)

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The Genetic Transformation ofChlamydia pneumoniae

mSphere ◽

10.1128/msphere.00412-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 9

Author(s):

Kensuke Shima ◽

Maximilian Wanker ◽

Rachel J. Skilton ◽

Lesley T. Cutcliffe ◽

Christiane Schnee ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

Fluorescent Protein ◽

Shuttle Vector ◽

Vector System ◽

Species Barrier ◽

Content Type ◽

Green Fluorescent ◽

Pathogenic Gene ◽

Plasmid Backbone

ABSTRACTWe demonstrate the genetic transformation ofChlamydia pneumoniaeusing a plasmid shuttle vector system which generates stable transformants. The equineC. pneumoniaeN16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearingC. pneumoniaetransformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolateC. pneumoniaeLPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolateC. pneumoniaeCV-6 and the human community-acquired pneumonia-associatedC. pneumoniaeIOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown thatChlamydiaspp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-freeC. trachomatis,C. muridarum,C. caviae,C. pecorum, andC. abortus. However, contrary to our expectation, pRSGFPCAT-Cpn did transformC. felis. Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid ofC. felis. Taken together, we provide for the first time an easy-to-handle transformation protocol forC. pneumoniaethat results in stable transformants. In addition, the vector can cross the species barrier toC. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCEThe absence of tools for the genetic manipulation ofC. pneumoniaehas hampered research into all aspects of its biology. In this study, we established a novel reproducible method forC. pneumoniaetransformation based on a plasmid shuttle vector system. We constructed aC. pneumoniaeplasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase inC. pneumoniae.C. pneumoniaetransformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation inC. pneumoniaeusing pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions inC. pneumoniaebiology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

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