1987 Fiscal Year Final Research Report Summary
Impoved electroporation system for high-vield gene transfer into mammalian cells.
| Project/Area Number |
61870099
|
|---|
| Research Category |
Grant-in-Aid for Developmental Scientific Research
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
医学一般
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
KATSRA Yoshimoto Chest Disease Research Institute, Kyoto University. Ptoseddor, 結核胸部疾患研究所, 教授 (90027095)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NISHIKAWA Shin-Ichi Chest Disease Research Institute, Kyoto University. Associate Professor, 結核胸部疾患研究所, 講師 (60127115)
OKADA Yasunobu Favulty of Maedicine, Kyoto University. Lecutrer, 医学部, 助教授 (10025661)
|
|---|
| Project Period (FY) |
1986 – 1987
|
| Keywords | Electroportion / Electric pulse / Gene / 遺伝子導入 |
|---|
| Research Abstract |
A variety of methods for DNA transfer, including protoplast fusion, DEAE dextran and calcium phosphate coprecipitation, are used in molecuar biology. The efficiency of transfection by these methods is often unacceptaby low in many cell species, especially in floating cells as well as normal diploid cells. Recentyl, a new transfection technique, electroporation, has been developed and reported to be effective for both adherent and floating cells.
So far, the capacity discharge method was employed to generate pulses which created membrane pores. However, this method has an inherent defect, which may interfer with reproducible experiments: The output impedance varis with frequency of pulses. Furthermore, this can deliver dacaying pulses but not trctangular pulses favorble for quantitative experiments.
To generate square pulses of high electric fields between electrode interposed with physiological electroloyte solutions of low electric resistances, a novel type of pulse generator with a ver
… More
y low output impedance (about 30 ohms) was constructed by using a pulse-transformation circuit in combination with a capacity discharge circuit. Both the pulse amplitude and duration can be quantitatively altered up to 30 KV and 300 <micrn>s, respectively. Plural pulses can be delivered at short intervals (whithin 1 s) without reduction of the amplitude.
Using this machine, efficiency of transfection of the plasmid DNA containing genes for the <mu>-chain of immunoglobulins was examined in moude myeloma J558L cells which express only the <iambda>-chain. The transformation frequency was found to increase with raising the pulse amplitude and dutaion. By applying two pulses of 3 KV/cm and 150 <micrn>s, high yield transfection (over 10^<-4>) was reproducebly attained. This electroporation technique was also applicable to human T-cell leukemia cells in which gene transfer has been unsuccessful with other techniques. Optimal conditions for gene transection in normal diploid cells could be investigated using this electroporation system. Less
|
