• Journal of Environmental Science International
• /
• v.32 no.3
• /
• pp.173-179
• /
• 2023

Rats are one of the most widely used animals in biomedical sciences because their metabolism and physiology are comparable to humans. In recent years, gene-targeted models have been developed using various animal species utilizing engineered nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas). It has recently become possible to efficiently transfect CRISPR/Cas into embryos via electroporation. However, electroporation can damage fertilized eggs; therefore, it is important to determine the optimal embryo culture conditions. A standardized approach for routine and reproducible rat transgenesis will render rat models more accessible for research. We performed experiments to obtain rat embryos with efficient superovulation and synchronization, and to investigate the appropriate medium conditions for pronuclear stage embryos subjected to electroporation stimulation for the introduction of engineered nuclease.

• Journal of Aquaculture
• /
• v.9 no.3
• /
• pp.293-300
• /
• 1996

Validities of several gene transfer methods including microinjection, electroporation and lipo-fection with luciferase gene (pRSVL), and effectiveness of mud loach expression vector which contains ARS from mud loach on production of transgenic mud loach were evaluated. Microiniection revealed the $0\~8\%$ of transgene incidence in 2-week-old fish with significant mosaicism. Electroporation and lipofection of mud loach sperm also successfully introduced the transgene into sperm cells, and transferred the foreign DNA into zygote. Gene transfer by electroporation and lipofection showed a range of $0\~28\%$ and $0\~48.1\%$ of transgene incidence, respectively in newly hatched larvae, altough most DNA introduced were gradually degraded with the development of fish. Microinjections of mud loach expression vector caused a significantly reduced survival rate of mud loach embryos with severe teratogenic effects, and ARS/Luc transgene could not be detected in normally developed fish after microinjection.

• Applied Biological Chemistry
• /
• v.36 no.6
• /
• pp.557-561
• /
• 1993

The neomycin phosphotransferase II gene (nptII) was introduced into geranium (Pelargonium zonale hybrids) protoplast by using PEG or electroporation method. The presence of the introduced DNA in the protoplast and the expressions of the gene in the transformed cells were examined. The presence of the nptII DNA in the protoplasts were detected by polymerase chain reaction. The expressions of nptII gene in the transformed cells were confirmed by the nptII assay.

• Mycobiology
• /
• v.38 no.4
• /
• pp.331-335
• /
• 2010

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/${\mu}g$ of DNA in $1{\times}10^7$ protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

• YAKHAK HOEJI
• /
• v.40 no.1
• /
• pp.59-64
• /
• 1996

Gram-positive bacteria, Bacillus subtilis BR151 and Staphylococcus aureus RN4220 were transformed with high efficiency by electroporation. The cells were incubated until late log phase, washed three times with 10% glycerol, 1mM HEPES, 12% sucrose and resuspended to $10^{10}{\sim}10^{11}cfu/ml$, then stored at -$70^{\circ}C$. Transformation efficiency of B. subtilis BR151 was $1.03{\times}10^7cfu/{\mu}g$ with cells washed with 10% glycerol and electroporated by 15KV/cm, 0.7msec pulse with pUB110. Transformation efficiency of S. aureus RN4220 was $4{\times}10^6cfu/{\mu}g$ with cells washed with 1mM HEPES + 10% glycerol and electroporated by 15KV/cm, 2.5msec pulse. The number of total transformants was 1000 when B. subtilis BRI51 was transformed with 100ng pUB110 DNA and the number of total transformants was 9000 when S. aureus RN4220 was transformed with 10ng pUB110 DNA

• Korean Journal of Plant Tissue Culture
• /
• v.21 no.4
• /
• pp.187-192
• /
• 1994

Wheat (Triticum aestium L.cv Cho-Kwang) explants were transformed by electrporation. Excised root segments were elechoporated with plasmid DNA of pBI121 and transferred to medium containing 300 mg/L kanamycin. Transformed calli formed within 5-7 days of culture and were selected from electroporated tissue on medium containing kanamycin after 4 weeks. The highest transformation frequency was obtained after electroporation with a pulse of 200 V/800 uF and calli formed at frequencies up to 2.5%. GUS ($\beta$-glucuronidase) assay and dot blot analysis showed that the foreign gene was capable of expressing in root explants subjected to electroporation and calli derived from the explants..

• Transactions of the Korean Society of Mechanical Engineers B
• /
• v.36 no.9
• /
• pp.901-905
• /
• 2012

We present an electroporation and viability monitoring chip for lung cancer cells in a single channel with multiple electric field zones. Previous electroporation chips utilized multiple microchannels or electrodes to form multiple electric fields, thus resulting in complex structures. However, the present chip can generate multiple electric fields in a single stepwise microchannel between a pair of electrodes, thus achieving the analysis of both cell electroporation and viability with a simple structure. We demonstrate that the electric field of 0.4 kV/cm results in a maximum percentage of $51.4{\pm}3.0%$ and $26.6{\pm}0.7%$ of viable and electroporated human lung cancer cells, H23 and A549, respectively. The present chip has potential for use in integrated cell chips for transfection studies.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.7
• /
• pp.947-959
• /
• 2012

This study was aimed at an evaluation of the potential inheritance of electroporation effects on Lactobacillus fermentum BT 8219 through to three subsequent subcultures, based on their growth, isoflavone bioconversion activities, and probiotic properties, in biotin-supplemented soymilk. Electroporation was seen to cause cell death immediately after treatment, followed by higher growth than the control during fermentation in biotin-soymilk (P<0.05). This was associated with enhanced intracellular and extracellular ${\beta}$-glucosidase specific activity, leading to increased bioconversion of isoflavone glucosides to aglycones (P<0.05). The growing characteristics, enzyme, and isoflavone bioconversion activities of the first, second, and third subcultures of treated cells in biotin-soymilk were similar to the control (P>0.05). Electroporation affected the probiotic properties of parent L. fermentum BT 8219, by reducing its tolerance towards acid (pH 2) and bile, lowering its inhibitory activities against selected pathogens, and reducing its ability for adhesion, when compared with the control (P<0.05). The first, second, and third subcultures of the treated cells showed comparable traits with that of the control (P>0.05), with the exception of their bile tolerance ability, which was inherited to the treated cells of the first and second subcultures (P<0.05). Our results suggest that electroporation could be used to increase the bioactivity of biotin-soymilk via fermentation with probiotic L. fermentum BT 8219, with a view towards the development of functional foods.

• Journal of Pharmaceutical Investigation
• /
• v.32 no.3
• /
• pp.181-184
• /
• 2002

This study reports the encapsulation of plasmid DNA in erythrocyte ghosts. The plasmid DNA was encapsulated into erythrocyte ghosts using three methods; osmotic shock, electroporation in isotonic medium, and e1ectroporation in hypotonic medium. Of three methods, electroporation in hypotonic medium resulted in the highest encapsulation efficiency of plasmid DNA. The morphology of erythrocyte ghosts prepared by electroporation in hypotonic medium was similar to that by osmotic shock alone. The circulation time of plasmid DNA in mice was prolonged by administration in erythrocyte ghost-encapsulated forms. These results indicated the potential of erythrocyte ghosts for biocompative nonviral delivery system of therapeutic genes for hematological diseases.

• 한국유가공학회:학술대회논문집
• /
• 1997.05a
• /
• pp.41-61
• /
• 1997

젖산균의 유전자 연구를 촉진하기 위해 간단하고 신속한 plasmid DNA의 분리방법과 electro-poration을 이용하여 vector plasmid의 간단하고 신속한 전이방법을 얻기 위해 젖산균의 형질전환에 영향하는 요인에 대하여 연구하였으며 연구결과는 다음과 같다. 1. O'Sullivan과 Klaenhammer의 방법을 개선하여 젖산균 plasmid DNA의 분리에 좋은 결과를 얻을 수 있는 신속하고 쉬운 방법을 고안하였으며, genomic DNA 분리에 이용되는 guanidium thio-cyanate 처리방법을 plasmid의 분리에 적용할 수 있었다. 2. L. casei, L. acidophilus. L. delbruekii var. bulgaricus. L. brevis와 L. plantarum 균주에서 plasmid를 확인하였으며, 돼지 분에서 분리된 L. lactis ssp. lactis. L. fermentum과 L. plantarum에서도 plasmid를 분리 확인하였다. 3. Lactococci의 plasmid분리는 lactobacilli와는 달리 mutanolysin의 처리없이도 잘 되었으며, L. lactis ssp. lactis와 Ent. faecalis에서 plasmid를 확인하였다. 4. E. coli plasimd 분리에 이용되는 MPS membrane filter 방법으로 젖산균 plasmid pLZ12의 분리가 가능하였으나, 세포파편이 filter를 막아 사용에 어려움이 있는 것으로 확인되었다. 5. Plasmid 분리없이 electroporation을 이용한 세포 대 세포 전이법으로 간편하고 빠르게 E. coli DH5${\alpha}$에 E. coli Jm109의 plasmid pBX19, pBR322를 전이시켰다. 6. L. lactis ssp. lactis 균주에 lysozyme 처리시 30${\sim}$80%의 생존율을 보였으며, 대부분의 L. acidophilus 균주의 경우 약 70%의 생존율을 보였다. L. casei 102S의 경우는 45분간 처리 시에도 100%의 생존율을 보였다. 8. L. lactis ssp. lactis 균주에 pLZ12를 6.0kV에서 전이시킨 결과 12.5kV에서보다 형질전환 효율이 훨씬 높았으며 lysozyme 처리에 의해 형질전환 효율이 증가되었다. 9. L. acidophilus 균주에 pLZ12를 전이시 6.0kV에서는 전이가 모두 이루어졌으나, 12.5kV에서는 L. acidophilus WIESBY와 NCFM에서 전이가 이루어지지 않았으며, lysozyme 처리 후 pLZ12를 전이시켰을 때 12kV보다 6.0kV에서 형질전환 효율이 증가되었다. 10. Gene Pulser와 Progenitor II를 사용하여 pLZ12를 L. lactis ssp. lactis 균주에 전이하였을 때 Gene Pulser에 비해 Progenitor II의 형질전환 효율이 현저히 떨어졌다. L. acidophilus HY7008과 HY7001은 두 기기 모두 형질전환이 이루어졌으나, L. acidophilus WEISBY와 NCFM은 Progeni-tor II에서 전이가 일어나지 않았으며, Gene Pulser에서 전이균주를 얻어 두 electroporator간에 형질전환 효율의 차이를 보였다. 11. L. casei 102S에 pLZ12를 electroporation시 낮은 전압에서 형질전환 효율이 비교적 좋았으며, 배양 시기를 달리하여 전이시켰을 때 대수생장 말기의 세포가 형질전환 효율이 좋았다. 12. L. casei 102S세포를 각각 10% glycerol, EB, 2차 증류수 등에 녹여 electroporation을 실시하였을 때 각각 $3.8{\times}10^3$, $5.0{\times}10^2$,1.5${\times}10^2$cfu의 형질전환 효율을 보였으며, 1.0mM HEPES, TE buffer를 사용하였을 때에는 전이가 이루어지지 않았다. 13. Plasmid pLZ12의 농도를 달리하여 electroporation을 하였을 때 형질전환 효율이 농도에 비례하여 증가하였다. 14. L. casei 102S에 대수생장 말기의 세포를 채취하여 10% glycerol, 200 Ohms, 25 ${\mu}$FD, 10kV/cm로 plasmid pLZ12를 electroporation할 때 최대 형질전환 효율인 3.8${\times}$10$^{3}$cfu를 얻었으며, lysozyme 처리가 다른 젖산균과는 달리 형질전환 효율을 증가시키지 못하였다. 15. L. casei 102S 세포를 10% glycerol과 EB에 녹여 -20$^{\circ}C$에서 냉동시킨 다음 1일과 7일 후의 세포를 electroporation한 결과 냉동시 세포에 손상을 주는 것으로 인식되었다.