Co-Epression of Two Transgenes in Lentiviral Vector Using Independent Transcriptional Units.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5254-5254
Author(s):  
Friedrich G. Schuening ◽  
Michail M. Zaboikin ◽  
Narasimhachar Srinivasakumar ◽  
Tatiana N. Zaboikina
Keyword(s):  
Flow Cytometry ◽  
Transgene Expression ◽  
Dna Methyltransferase ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Egfp Expression ◽  
Drug Resistant ◽  
Gfp Expression ◽  
Ic50 Values ◽  
Hiv 1

Abstract There are several gene therapy approaches, which require transfer and co-expression of two transgenes within one target cell. To this end, we have created and tested two-gene expression HIV-1 based vectors, which encode enhanced green fluorescent protein (EGFP) and P144K mutant of canine O6-methylguanine-DNA-methyltransferase (MGMT) transgenes under either the phosphoglycerate kinase gene promoter (PGKp), or the elongation factor 1 alpha promoter (EF1a_p). Eight different configurations of the two transgene expression cassettes were created and tested within the same lentiviral backbone (see Figure). Individual VSV-G pseudotyped vectors stocks were produced and used for infection of canine thyroid adenocarcinoma (CTAC) cells at low multiplicity of infection (MOI = 0.1) to ensure 1 copy of proviral vector per transduced cell. The cells were harvested one week later and an aliquot was assayed for EGFP expression by flow cytometry. Another portion was subjected to selection with O6-benzylguanine (BG, 40 m M for 18 hrs) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 100 m M for 2 hrs) and kept in culture for additional two weeks to eliminate cells expressing insufficient MGMT. The percentage of GFP positive cells, prior to selection with BG and BCNU, ranged between 0.1 % to 6.6% for the dual-transgene expression cassette encoding HIV-1 vectors. Following selection with BG and BCNU, the percentage of GFP positive cells increased for all vectors with the exception of vector #2 PGEM. Two of the vectors (#1 PMEG and #8 EGMP) demonstrated over 80% GFP positivity after selection. The results of flow cytometry after selection were corroborated by fluorescence microscopy of individual BCNU-resistant colonies. GFP expression was readily detected in drug-resistant colonies transduced with vectors #1 PMEG or #8 EGMP. Weaker GFP expression was detected in drug resistant cells transduced with vectors #3 PMGE, #5 EMPG or #6 EGPM. No significant GFP expression was observed in drug-resistant colonies transduced with vectors #2 PGEM, #4 PGME or #7 EMGP. Drug resistance to BCNU (IC50 values) provided by each of the vectors, was also determined. The data showed that the IC50 values for #1 PMEG and #8 EGMP vectors were 2.4-fold and 4.4-fold, respectively, higher than for mock transduced control cells. The above results indicate that coordinated co-expression of two transgenes using independent expression cassettes is promoter, position and orientation dependent. The data also indicate that two potentially useful vectors (#1 PMEG and #8 EGMP) have been identified for evaluation, ex vivo and in vivo, in the canine model for co-expression of two transgenes.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 232
Author(s):  
L. N. Moro ◽  
G. Vichera ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Egfp Expression ◽  
Bovine Embryos ◽  
Exogenous Dna ◽  
Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

Efficient transgenic rat production by a lentiviral vector

2007 ◽  
Vol 293 (1) ◽  
pp. H881-H894 ◽  
Author(s):  
Mieczyslaw Michalkiewicz ◽  
Teresa Michalkiewicz ◽  
Aron M. Geurts ◽  
Richard J. Roman ◽  
Glenn R. Slocum ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Insertion Site ◽  
Single Copy ◽  
Egfp Expression ◽  
Transgenic Rat ◽  
Sprague Dawley ◽  
Lentiviral Transgenesis ◽  
Sprague Dawley Rats ◽  
Transgenic Lines

A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken β-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit β-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and ∼100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F1population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F1transgenic lines, indicating that the expression of the transgene was independent of its chromosomal position. Thus lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and will enhance the usefulness of this species in gene discovery and target validation studies.

Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5484-5484
Author(s):  
Yuan Lin ◽  
Stanton L. Gerson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Lentiviral Vector ◽  
Bioluminescent Imaging ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

Chicken HS4 Insulators Have Minimal Functions in Human Hematopoietic Cells That Were Transduced with An HIV1-Based Lentiviral Vector.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3570-3570
Author(s):  
Naoya Uchida ◽  
Kareem Washington ◽  
Matthew M. Hsieh ◽  
John F. Tisdale
Keyword(s):  
Transgene Expression ◽  
Lentiviral Vector ◽  
Globin Gene ◽  
Hematopoietic Cells ◽  
Erythroid Cells ◽  
Gfp Expression ◽  
Position Effects ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Insulator Elements

Abstract Abstract 3570 Poster Board III-507 For the hemoglobin disorders, hematopoietic stem cell gene transfer is potentially curative, yet this strategy requires high-level β-globin gene expression among erythroid cells. Position effects, which are imparted by chromosomal position and chromatin structure, induce clonal variability of transgene expression. Recent work demonstrates that the chicken HS4 insulator element reduces position effects, resulting in consistently high-level expression of a therapeutic β-globin gene in the MEL cell line. In this study, we evaluated the effects of HS4 insulators on lentiviral vector titers and transgene expression among transduced human hematopoietic cells. We constructed various types of insulated lentiviral vectors using a reverse oriented GFP under the control of the MSCV-LTR promoter (rMpGFP) or a conventional reverse oriented β-globin expression cassette, in which the globin gene was changed to GFP (BGpGFP). A full-length HS4 insulator (1.2 kb HS4), tandem HS4 core insulator (2 × 250 b HS4), and a single core insulator (250 b HS4) were inserted into the 3′ LTR. The insulator elements were inserted in both forward (F) and reverse (R) orientations. Vector titers were significantly decreased by insertion of the 1.2 kb HS4 and 2 × 250 b HS4 in both orientations and both vector constructs, compared to uninsulated vectors (p<0.05), with the degree dependent on fragment size. Interestingly, reverse-oriented insulators showed better vector titers when compared to forward-oriented insulators for all types of insulator fragments except the 2 × 250 b HS4 in rMpGFP vectors (p<0.05). We next evaluated GFP expression from various insulated rMpGFP vectors in GPA+ human erythroid cells that originated from transduced CD34+ cells (MOI=3) (Figure). The %GFP was decreased by 1.2 kb HS4 and 2 × 250 b HS4 insulators in both orientations, compared to the uninsulated vector (p<0.05). All insulated vector constructs had a tendency to lower CVs, there was no significant difference except for the 1.2 kb HS4 F vector (p<0.05). There was no significant difference of MFIs between all types of insulated and uninsulated vectors. In order to evaluate insulator function for the BGpGFP vectors in human hematopoietic cells, we practically chose the 250 b HS4 R because it did not decrease vector titers and the 1.2 kb HS4 showed 5-fold lower transduction efficiency in human erythroid cells. During erythorid culture of transduced human erythroid cells, %GFP and MFIs decreased whereas CVs increased,showing chromosomal position effects. The 250 b HS4 R insulator showed lower %GFP and lower MFIs (MOI=20) (p<0.05 on day 13 and 20), compared to those of the uninsulated vector. There was no significant difference in CVs. After MOI escalation of BGpGFP vectors (day13), the insulated vector showed lower %GFP at MOI 10, 25, and 50 (p<0.05) and lower overall GFP expression (%GFP x MFI) at MOI 25 and 50 (p<0.05) compared to uninsulated vector. These data demonstrated that inclusion of HS4 insulator elements decreases GFP expression, which is not overcome by increasing MOI. We then performed transduced hematopoietic stem cell transplantation in a human xenograft mouse model using a 250 b HS4 R insulated rMpGFP vector. In the human CD45+ fraction of mouse peripheral blood cells, the insulator element decreased both %GFP and MFIs at 4 and 8 weeks after transplantation (p<0.05). There was no significant difference of CVs among the insulated and uninsulated vector at all time points. These data demonstrate that the inclusion of HS4 insulator elements lowers viral titers, reduces efficiency of transduction and produces minimal effects on transgene expression among human hematopoietic cells in vitro and in vivo Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene Therapy Applications of Non-Human Lentiviral Vectors

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1106
Author(s):  
Altar M. Munis
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Cell Therapies ◽  
Dividing Cells ◽  
Cell Genome ◽  
Hiv 1

Recent commercialization of lentiviral vector (LV)-based cell therapies and successful reports of clinical studies have demonstrated the untapped potential of LVs to treat diseases and benefit patients. LVs hold notable and inherent advantages over other gene transfer agents based on their ability to transduce non-dividing cells, permanently transform target cell genome, and allow stable, long-term transgene expression. LV systems based on non-human lentiviruses are attractive alternatives to conventional HIV-1-based LVs due to their lack of pathogenicity in humans. This article reviews non-human lentiviruses and highlights their unique characteristics regarding virology and molecular biology. The LV systems developed based on these lentiviruses, as well as their successes and shortcomings, are also discussed. As the field of gene therapy is advancing rapidly, the use of LVs uncovers further challenges and possibilities. Advances in virology and an improved understanding of lentiviral biology will aid in the creation of recombinant viral vector variants suitable for translational applications from a variety of lentiviruses.

Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

2016 ◽  
Vol 26 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Lin Liu ◽  
Xiangyan Zhang ◽  
Siyuan Yang ◽  
Yao Zhai ◽  
Weijia Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Qrt Pcr ◽  
Key Factor ◽  
Green Fluorescent

<b><i>Aims:</i></b> The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. <b><i>Methods:</i></b> The <i>bla</i><sub>CTX-M</sub> promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of <i>bla</i><sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. <b><i>Results:</i></b> The promoters of all the <i>bla</i><sub>CTX-M</sub> genes were provided by IS<i>Ecp1 </i>and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of <i>bla</i><sub>CTX-M</sub> was influenced. <b><i>Conclusion:</i></b> The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the <i>bla</i><sub>CTX-M</sub> gene does not specifically enhance the expression of<i> bla</i><sub>CTX-M</sub>, contrary to previous reports. The expression of <i>bla</i><sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.

scholarly journals Enhancement of β-Globin Locus Control Region-Mediated Transactivation by Mitogen-Activated Protein Kinases through Stochastic and Graded Mechanisms

1999 ◽  
Vol 19 (8) ◽  
pp. 5565-5575 ◽  
Author(s):  
E. Camilla Forsberg ◽  
Tatiana N. Zaboikina ◽  
Wayne K. Versaw ◽  
Natalie G. Ahn ◽  
Emery H. Bresnick
Keyword(s):  
Flow Cytometry ◽  
Control Region ◽  
Fluorescent Protein ◽  
Mapk Pathway ◽  
Locus Control Region ◽  
Mitogen Activated Protein Kinase ◽  
Egfp Expression ◽  
Enhancer Activity ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

ABSTRACT Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the β-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756–8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Aγ-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

scholarly journals Creation and characterisation of 11β hydroxysteroid dehydrogenase type 1 transgenic mesenchymal stem cells for use in models of acute respiratory distress syndrome

2019 ◽  
Author(s):  
RAHUL Y. MAHIDA ◽  
Zhengqiang Yuan ◽  
Krishna K. Kolluri ◽  
Sebastian T. Lugg ◽  
Aaron Scott ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Respiratory Distress Syndrome ◽  
Stem Cell ◽  
Transgene Expression ◽  
Distress Syndrome ◽  
Lentiviral Vector ◽  
Transfection Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Alveolar Space

Abstract Background: Human bone marrow mesenchymal stem cell (MSC) administration reduces inflammation in pre-clinical models of lung injury, however clinical efficacy in patients with acute respiratory distress syndrome (ARDS) has not been shown. Upregulation of the glucocorticoid activating enzyme, 11β hydroxysteroid dehydrogenase type 1 (HSD-1) within the alveolar space elevates local anti-inflammatory cortisol levels, and promotes alveolar macrophage efferocytosis. Administration of HSD-1 transgenic MSCs (tMSCs) that overexpress HSD-1 may enhance local cortisol activation. This combined cellular and gene therapy may be more effective at reducing inflammation in ARDS than cellular therapy alone. Methods: Molecular cloning was used to create a recombinant lentiviral vector containing the HSD-1 and green fluorescent protein (GFP) transgenes. MSCs were transfected using this lentivirus, then transfection efficiency was assessed using flow-cytometry. HSD-1 transgene expression was assessed using immunofluorescence and western blotting. Thin layer chromatography was used to assess HSD-1 function in tMSCs. Bi-lineage differentiation and flow-cytometry were used to determine whether tMSCs maintained a stem cell phenotype. Results: A recombinant lentiviral vector was created containing the HSD-1 and GFP transgenes under the control of a tetracycline promoter. MSCs were successfully transfected, with a transfection efficiency of 91.1%. HSD-1 transgene expression was confirmed by immunofluorescence and western blot. Functional HSD-1 activity was evident within tMSCs, with predominant reductase cortisol activation, following treatment with the transcriptional activator doxycycline. HSD-1 reductase activity was maintained for 72 hours after doxycycline was removed from tMSCs. HSD-1 tMSCs maintained the capacity for osteogenic and adipogenic differentiation, and maintained expression of MSC surface markers. Conclusions: We successfully transduced tMSCs to express the HSD-1 transgene that functions predominantly as a reductase. Administration of HSD-1 tMSCs to in vivo models of ARDS may attenuate inflammation through activation of cortisol in the alveolar space.

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