Gene transfer into CD4+ T lymphocytes: Green fluorescent protein-engineered, encephalitogenic T cells illuminate brain autoimmune responses

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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Imaging Changes in Lymphoid Organs In Vivo after Brain Ischemia with Three-Dimensional Fluorescence Molecular Tomography in Transgenic Mice Expressing Green Fluorescent Protein in T Lymphocytes

Molecular Imaging ◽

10.2310/7290.2008.00016 ◽

2008 ◽

Vol 7 (4) ◽

pp. 7290.2008.00016 ◽

Cited By ~ 25

Author(s):

Abraham Martin ◽

Juan Aguirre ◽

Ana Sarasa-Renedo ◽

Debbie Tsoukatou ◽

Anikitos Garofalakis ◽

...

Keyword(s):

Green Fluorescent Protein ◽

T Lymphocytes ◽

Transgenic Mice ◽

Brain Ischemia ◽

Fluorescent Protein ◽

Three Dimensional ◽

Lymphoid Organs ◽

Fluorescence Molecular Tomography ◽

Green Fluorescent

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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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