Gene transfer by protoplast fusion: Expression cloning of rare gene products in mammalian cells

1993 ◽  
Vol 15 (2) ◽  
pp. 69-71
Author(s):  
Lynn Helena Caporale ◽  
Patricia DeHaven
Keyword(s):  
Gene Transfer ◽  
Protoplast Fusion ◽  
Mammalian Cells ◽  
Fusion Expression ◽  
Expression Cloning ◽  
Gene Products ◽  
Rare Gene

Protoplast fusion in microtiter plates for expression cloning in mammalian cells: demonstration of feasibility using membrane-bound alkaline phosphatase as a reporter enzyme

Gene ◽  
1990 ◽  
Vol 87 (2) ◽  
pp. 285-289 ◽  
Author(s):  
Lynn H. Caporale ◽  
Nicole Chartrain ◽  
Michael Tocci ◽  
Patricia DeHaven
Keyword(s):  
Alkaline Phosphatase ◽  
Protoplast Fusion ◽  
Mammalian Cells ◽  
Expression Cloning ◽  
Microtiter Plates ◽  
Reporter Enzyme ◽  
Membrane Bound

scholarly journals CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 692
Author(s):  
Sweta Talyan ◽  
Samantha Filipów ◽  
Michael Ignarski ◽  
Magdalena Smieszek ◽  
He Chen ◽  
...  
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
De Novo ◽  
Depth Information ◽  
Gene Products ◽  
Classical Analysis ◽  
Protein Coding ◽  
Bioinformatic Pipeline ◽  
Non Coding Rnas ◽  
Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

Current Questions of Gene Transfer via Protoplast Fusion in Microorganisms

1983 ◽  
pp. 137-142
Author(s):  
L. Ferenczy
Keyword(s):  
Gene Transfer ◽  
Protoplast Fusion

scholarly journals Baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells

1999 ◽  
Vol 1 (2) ◽  
pp. 93-102 ◽  
Author(s):  
G. Duisit ◽  
S. Saleun ◽  
S. Douthe ◽  
J. Barsoum ◽  
G. Chadeuf ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Heparan Sulfate ◽  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Baculovirus Vector ◽  
Efficient Gene

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion

1981 ◽  
Vol 1 (8) ◽  
pp. 743-752
Author(s):  
R M Sandri-Goldin ◽  
A L Goldin ◽  
M Levine ◽  
J C Glorioso
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protoplast Fusion ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
High Frequency ◽  
Mammalian Cells ◽  
Frequency Transfer ◽  
Simplex Virus

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.

Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells

1982 ◽  
Vol 2 (6) ◽  
pp. 607-616
Author(s):  
Masahiro Ishiura ◽  
Susumu Hirose ◽  
Tsuyoshi Uchida ◽  
Yoshio Hamada ◽  
Yoshiaki Suzuki ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Gene Transfer ◽  
Mammalian Cells ◽  
Phage Particle ◽  
High Efficiency ◽  
Linear Increase ◽  
Recombinant Phage ◽  
Optimal Values ◽  
Simplex Virus

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk − cells to the TK + phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25°C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10 5 phage particles per 10 6 cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 × 10 7 phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10 10 phage particles per dish, did not significantly affect the number of transformants.

In vitro gene transfer in mammalian cells via a new cationic liposome formulation.

1998 ◽  
Author(s):  
M C Kao ◽  
S L Law ◽  
T C Chuang ◽  
Y S Lin
Keyword(s):  
Gene Transfer ◽  
Mammalian Cells ◽  
Cationic Liposome ◽  
Liposome Formulation

Surfactant-Mediated Gene Transfer for Mammalian Cells

1997 ◽  
pp. 381-385
Author(s):  
Masamichi Kamihira ◽  
Jun You ◽  
Shinji Iijima
Keyword(s):  
Gene Transfer ◽  
Mammalian Cells
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