197 INFLUENCE OF PLASMID CONSTRUCTION AND CELL TYPE ON THE GENERATION OF GENETICALLY MODIFIED CELL LINES IN CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 189
Author(s):  
A. G. Curcio ◽  
F. F. Bressan ◽  
K. S. Viana ◽  
A. F. L. Rios ◽  
F. V. Meirelles ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Production Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Specific Cell ◽  
Cell Type ◽  
Transfected Cells ◽  
Plasmid Construction ◽  
Fetal Fibroblasts ◽  
Adult Fibroblasts

Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN2 plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP+ cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student–Newman–Keuls test (SNK; P < 0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65, CC: 3.9 ± 1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542 ± 497.09; AF: 9367.5 ± 3490.9, CC: 3496 ± 2638.92). The pEGFPN2 plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 – FF: 4.9 ± 0.14 and 206.47 ± 755; AF: 760 and 2.4 ± 0.70 ± 330.92; CC: 3.9 ± 1.97, and 1418 ± 36.06, respectively; control – FF: 0.15 ± 0.07 and 249 ± 6 : 36; AF: 0.15 ± 0.07 588 ± 213.54, and CC 0.05 ± 0 214 ± 0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.

scholarly journals Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method

2019 ◽  
Vol 32 (1) ◽  
pp. 34-42
Author(s):  
Alinne G Curcio ◽  
Fabiana F Bressan ◽  
Carla S Paes De Carvalho ◽  
Célia R Quirino ◽  
Flavio V Meirelles ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescence Intensity ◽  
Transgene Expression ◽  
Cumulus Cells ◽  
Cell Types ◽  
Cell Type ◽  
Lentiviral Transduction ◽  
Transfer Method ◽  
Gene Transfer Method ◽  
Adult Fibroblasts

Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.Keywords: cloning, epigenetics, lipofection, lentiviral transduction, nuclear reprogramming.  Resumen Antecedentes: La producción de animalestransgénicossigue siendo una biotecnología de baja eficiencia, y se deberían utilizar alternativas sencillas para mejorar la tasa de producción de bovinos transgénicos mediante transferencia nuclear. Una de estas alternativas es la selección del tipo mas apropiado de célula donante y método de transferencia génica. Objetivo: Investigar el efecto del tipo celular (fibroblastos fetales o adultos, y celulas del cumulus), y el método de transferencia génica (lipofección y transducción lentiviral) en la incorporación, expresión génica, y la intensidad de fluorescencia del transgén en células bovinas analizadas por citometría de flujo. Métodos: Fibroblastos fetales (FF), fibroblastos adultos (AF), y células del cúmulo (CC) fueron transfectados a través de lipofección o transducidos utilizando partículas lentivirales producidas con plásmidos que expresan la proteína verde fluorescente (GFP). Resultados: La transducción lentiviral dio lugar a mayores tasas de expresión del transgen en todos los tipos de células (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96, CC: 60,7% ± 14,7) en comparación con la lipofección (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). Las células del cúmulus mostraron menores tasas de expresión del transgen que los otros tipos celulares. En cuanto a la intensidad de fluorescencia, no hubo diferencias significativas entre lipofección y transducción lentiviral; en ambos tratamientos, se obtuvo una mayor intensidad de fluorescencia cuando se usaron células adultas en lugar de células fetales. Conclusión: La eficiencia de la transferencia de genes varía según el tipo de célula y el método de transferencia génica, con la transducción lentiviral se logra una mayor tasa de transfección, y los fibroblastos adultos muestran una mejor expresión transgénica.Palabras clave: clonación, epigenética, lipofección, reprogramación nuclear, transducción lentiviral.  Resumo Antecedentes: A produção de animais transgênicos é uma biotecnologia que ainda apresenta baixa eficiência e alternativas simples devem ser usadas para o aumento da produção de bovinos transgênicos por transferência nuclear. Uma destas alternativas compreende a seleção do tipo apropriado de célula doadora de núcleo e do método de transferência gênica. Objetivo: Investigar a influência do tipo celular (fibroblastos fetais ou adultos, e células do cumulus), e do método de transferência gênica (transfecção por lipofecção ou transdução lentiviral) na incorporação, expressão, e na intensidade de fluorescência do transgene em células bovinas analisadas por citometria de fluxo. Métodos: Fibroblastos fetais (FF), fibroblastos adultos (AF), e células do cumulus (CC) foram submetidas à lipofecção ou à transfecção lentiviral utilizando plasmídeos expressando a Proteína Fluorescente Verde – GFP). Resultados: A transdução lentiviral resultou em maiores taxas de expressão do transgene em todos os tipos celulares (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96; CC: 60,7% ± 14.7) quando comparada com a lipofeccção (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). As células do cumulus apresentaram menores taxas de expressão quando comparadas aos outros tipos celulares. Com relação à intensidade de fluorescência, não houve diferença significativa entre a lipofecção e a transdução lentiviral e em ambos os tratamentos as células adultas apresentaram maior intensidade de fluorescência do que as células fetais. Conclusão: A eficiência de transferência gênica varia de acordo com o tipo celular, e com o método de transferência gênica, sendo que a transdução lentiviral resultou em maiores taxas, e que os fibroblastos adultos mostraram melhor expressão do transgene.Palavras-chave: clonagem, epigenética, lipofecção, reprogramação nuclear, transdução lentiviral.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

56 TRANSGENESIS BY HANDMADE CLONING USING EGFP-TRANSFECTED YUCATAN FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
P. M. Kragh ◽  
Y. Du ◽  
J. Li ◽  
Y. Zhang ◽  
L. Bolund ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cumulus Cells ◽  
Miniature Pig ◽  
Egfp Gene ◽  
Fusion Procedure ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, genetic manipulations can be performed in cells isolated from special breeds followed by SCNT into enucleated oocytes isolated from slaughterhouse ovaries. In the present study, we established production of Yucatan blastocysts by the handmade cloning (HMC) technique using non-transgenic fibroblasts from the Yucatan miniature pig, and produced transgenic blastocysts using enhanced green fluorescent protein (EGFP)-positive Yucatan fetal fibroblasts. For transgenesis, Yucatan fibroblasts from a 40-day old fetus were transfected with a vector containing an EGFP gene and a neomycin-resistance selection gene by lipofection. Well separated neomycin-resistant colonies were isolated, expanded, and used for HMC. For HMC, cumulus–oocyte complexes were aspirated from ovaries of slaughterhouse sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The remaining parts, i.e. cytoplasts, were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused with one fibroblast in the absence of Ca2+. After one h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, and subsequently cultured in cytochalasin B and cycloheximide for 4 h. The development of reconstructed embryos to the blastocyst stage was determined after 7 days of in vitro culture. When using non-transgenic and EGFP-positive Yucatan fetal fibroblasts, the rate of blastocyst formation (mean � SEM) were 36 � 7% (36/102) and 42 � 7% (32/77), respectively. In conclusion, the HMC technique was very efficient for production of blastocysts of Yucatan miniature pig origin using both non-transgenic and EGFP-positive fetal fibroblasts.

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 151-158 ◽  
Author(s):  
Rita P. Cervera ◽  
Fernando García-Ximénez
Keyword(s):  
Technical Efficiency ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cell Types ◽  
Cell Type ◽  
Fetal Fibroblasts ◽  
Somatic Nuclear Transfer ◽  
Metaphase Ii Oocytes

The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion rate was obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.

scholarly journals 46 EFFICIENT TRANSFECTION OF PLASMID DNA INTO CELLS FOR USE AS NUCLEAR DONORS

2005 ◽  
Vol 17 (2) ◽  
pp. 172
Author(s):  
S.-L. Lee ◽  
S.-A. Ock ◽  
H.-J. Song ◽  
B. Mohana Kumar ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
South Korea ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Cumulus Cells ◽  
Cell Types ◽  
Development Project ◽  
Exogenous Dna ◽  
Transfected Cells ◽  
Fetal Fibroblasts

Somatic cell nuclear transfer (SCNT) has the potential to significantly improve the production of valuable livestock that produce recombinant proteins, such as pharmaceutical proteins for human disease or biomaterials for medical use. The success of this potential depends on efficient and optimized protocols for introducing exogenous DNA into cells. In this study, we compared two methods of transfection, Effectene (Qiagen, Inc., Valencia, CA, USA) and electroporation. Plasmid DNA (pEGFF-N1, Clontech, Seoul, Korea) was transfected into fetal fibroblasts (FFB), cumulus cells (CUC), and adult ear skin cells (ESC). Transfection efficiency, chromosome normality, gene expression, and apoptosis were assessed. Cells cultured in α-modified Eagle's medium (α-MEM; BioWhittaker, Walkersville, MD, USA) + 10% FBS were transfected with pEGFP-N1. For electroporation, cells (5 × 106 cells/mL) were mixed in 300 μL perrim buffer (75% Cytosalts with 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 5 mM MgCl2, and 25% α-MEM) + 15 μg pEGFP-N1, and subjected to two pulses of 0.38 kV and 400 μF delivered by Gene Pulser (Bio-Rad; BMS, Ltd., Seoul, South Korea). For Effectene transfection, the procedure suggested by the manufacture was followed. Transfected cells were selected with 600 μg/mL G418 (Gibco; KDR Biotech Co., Ltd., Seoul, South Korea) and cultured at 39°C, 5% CO2 in air. Assessments of EGFP transfected cells by green fluorescence was carried out under an inverted epifluorescence microscope (Nicon, Kanagawa, Japan) equipped with a filter for FITC (excitation maximum = 488 nm; emission maximum = 507 nm). Differences among the groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Most cells (>80%) in confluence were at G0/G1 phase, and transfection of the gene into all three cell types did not affect the incidence of chromosomal abnormality or change morphology. In addition, the rates of apoptosis assessed by TUNEL did not differ in all three cell types by either method of transfection at different cell passages. However, the efficiency of gene transfection into FFB by Effectene reagent (14.2 ± 1.7%) was significantly (P < 0.05) higher than that by electroporation (5.1 ± 1.0%). Among the three type cells, the efficiency of gene transfection by Effectene and electroporation of FFB (14.2 ± 1.7 and 5.1 ± 1.0%, respectively) was significantly (P < 0.05) higher than those of CUC and ESC (9.4 ± 1.5 and 3.3 ± 0.8; 8.8 ± 0.7 and 2.1 ± 0.4%, respectively). In conclusion, although there were no differences in the alteration of chromosomes, cell morphology, and apoptosis among three cell types transfected with or without plasmid DNA, FFB is the most effective cell type to be transfected. Effectene is superior to other currently available methods for introducing plasmid DNA into a variety of cells. The high level of transfection achieved by Effectene will encourage its use as a tool for producing transgenic embryos and animals by SCNT. This work was supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010.

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 67-72 ◽  
Author(s):  
So Gun Hong ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
Min Jung Kim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Transfected Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

scholarly journals Epigenetic reprogramming of cell identity: lessons from development for regenerative medicine

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Amitava Basu ◽  
Vijay K. Tiwari
Keyword(s):  
Regenerative Medicine ◽  
Cell Types ◽  
Direct Conversion ◽  
Cellular Reprogramming ◽  
Specific Cell ◽  
Cell Type ◽  
Pathological Conditions ◽  
Gene Regulatory ◽  
Induced Pluripotent ◽  
And Function

AbstractEpigenetic mechanisms are known to define cell-type identity and function. Hence, reprogramming of one cell type into another essentially requires a rewiring of the underlying epigenome. Cellular reprogramming can convert somatic cells to induced pluripotent stem cells (iPSCs) that can be directed to differentiate to specific cell types. Trans-differentiation or direct reprogramming, on the other hand, involves the direct conversion of one cell type into another. In this review, we highlight how gene regulatory mechanisms identified to be critical for developmental processes were successfully used for cellular reprogramming of various cell types. We also discuss how the therapeutic use of the reprogrammed cells is beginning to revolutionize the field of regenerative medicine particularly in the repair and regeneration of damaged tissue and organs arising from pathological conditions or accidents. Lastly, we highlight some key challenges hindering the application of cellular reprogramming for therapeutic purposes.

scholarly journals Shisa6 mediates cell-type specific regulation of depression in the nucleus accumbens

2021 ◽  
Author(s):  
Hee-Dae Kim ◽  
Jing Wei ◽  
Tanessa Call ◽  
Nicole Teru Quintus ◽  
Alexander J. Summers ◽  
...  
Keyword(s):  
Nucleus Accumbens ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Current Treatment ◽  
Specific Cell ◽  
Excitatory Synapses ◽  
Cell Type ◽  
Cell Type Specific ◽  
Specific Regulation ◽  
Circuit Function

AbstractDepression is the leading cause of disability and produces enormous health and economic burdens. Current treatment approaches for depression are largely ineffective and leave more than 50% of patients symptomatic, mainly because of non-selective and broad action of antidepressants. Thus, there is an urgent need to design and develop novel therapeutics to treat depression. Given the heterogeneity and complexity of the brain, identification of molecular mechanisms within specific cell-types responsible for producing depression-like behaviors will advance development of therapies. In the reward circuitry, the nucleus accumbens (NAc) is a key brain region of depression pathophysiology, possibly based on differential activity of D1- or D2- medium spiny neurons (MSNs). Here we report a circuit- and cell-type specific molecular target for depression, Shisa6, recently defined as an AMPAR component, which is increased only in D1-MSNs in the NAc of susceptible mice. Using the Ribotag approach, we dissected the transcriptional profile of D1- and D2-MSNs by RNA sequencing following a mouse model of depression, chronic social defeat stress (CSDS). Bioinformatic analyses identified cell-type specific genes that may contribute to the pathogenesis of depression, including Shisa6. We found selective optogenetic activation of the ventral tegmental area (VTA) to NAc circuit increases Shisa6 expression in D1-MSNs. Shisa6 is specifically located in excitatory synapses of D1-MSNs and increases excitability of neurons, which promotes anxiety- and depression-like behaviors in mice. Cell-type and circuit-specific action of Shisa6, which directly modulates excitatory synapses that convey aversive information, identifies the protein as a potential rapid-antidepressant target for aberrant circuit function in depression.

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.

Sign in / Sign up

Export Citation Format

Share Document