Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization.
نویسندگان
چکیده
Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 microsecond) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV/cm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma cell lines for use in subsequent fusion experiments.
منابع مشابه
Effective in vitro gene delivery to murine cancerous brain cells using carbon nanotube-polyethylenimine conjugates
Objective(s): Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In t...
متن کاملPreparation, characterization and transfection efficiency of nanoparticles composed of alkane-modified polyallylamine
Objective(s): Although viral vectors are considered efficient gene transfer agents, their board application has been limited by toxicity, immunogenicity, mutagenicity and small gene carrying capacity. Non-viral vectors are safe but they suffer from low transfection efficiency. In the present study, polyallylamine (PAA) in two molecular weights (15 and 65 kDa) was modified by alkane derivatives ...
متن کاملTHE PRODUCTION OF MURINE MONOCLONAL ANTIBODIES (MAb) DIRECTED AGAINST HUMAN T- LYMPHOCYTE SUBSETS
The production of murine monoclonal antibody (MAb) has not yet been reported in Iran. The present work describes for the first time the generation of several murine hybridoma clones secreting MAbs directed against human leukocyte surface antigens. The secreted antibodies by hybridoma clones have been screened on different lymphoid and non-lymphoid tissues. Results indicated that of seven h...
متن کاملLow-frequency ac electroporation shows strong frequency dependence and yields comparable transfection results to dc electroporation.
Conventional electroporation has been conducted by employing short direct current (dc) pulses for delivery of macromolecules such as DNA into cells. The use of alternating current (ac) field for electroporation has mostly been explored in the frequency range of 10kHz-1MHz. Based on Schwan equation, it was thought that with low ac frequencies (10Hz-10kHz), the transmembrane potential does not va...
متن کاملOptimization of SW480 Colon Cancer Cells Transfection with Lipofectamine 2000
ABSTRACT Background and Objectives: Nonviral carriers including those based on synthetic cationic lipids, offer several advantages over the viral counterparts. These carriers are able to form complexes with nucleic acids and deliver genes into the cells via the cellular endocytosis pathway, without significant toxicity. The level of transg...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Journal of immunological methods
دوره 109 1 شماره
صفحات -
تاریخ انتشار 1988