I would like to introduce large size plasmid vector (12kb) into chick embryo by in ovo electroporation. However, the number of electroporated cells, which are indicated by fused fluorescent tag, is significantly low as compered to small plasmids under ~8kb. The expression vector is pCAGGS (4.7kb) and aimed cDNA size is 7.5kb.
Can you provide some details on what electroporation parameters you are using. We have some experience with transfection of large (15kb) plasmids into cancer cells using electroporation. In our experience you can significantly improved results by adjusting to a single short high voltage pulse (1000v/cm - 100usec) followed by a lower voltage -(between 25-100v/cm) and longer pulse duration (e.g. 10msec). Can require some optimisation depending on the cell line.
Declan Soden, CCRC, Cork, Ireland
do you have access to a nucleofector (Lonza)? this can significantly enhance transfection efficiency in difficult to transfect cells - although its possible you may struggle either way with such a large plasmid with toxicity etc.
Can you provide some details on what electroporation parameters you are using. We have some experience with transfection of large (15kb) plasmids into cancer cells using electroporation. In our experience you can significantly improved results by adjusting to a single short high voltage pulse (1000v/cm - 100usec) followed by a lower voltage -(between 25-100v/cm) and longer pulse duration (e.g. 10msec). Can require some optimisation depending on the cell line.
Declan Soden, CCRC, Cork, Ireland
Can you also provide information on which tissue you are electroporating into the chick embryo and what conditions you are using i.e. Voltage, pulses etc.
Many thanks for your reply.
Nucleofector is available in our institute but aimed target is chick embryo but cultured tissue. I didn't know that Nucleofector is effective in large plasmid transfection. If an opportunity offers I want to test that in cultured chick cells.
My electroporator is CUY21 EX pulse generator (BEX inc.). This electroporator can generate short pulse and long pulses, sequentially. Parameter of electroporation is 13V short pulse for 0.08 sec once followed by 5-6V long pulses for 50msec, 3 times. This config has been used successfully for transfection of usual size plasmid. I would like to target prospective paraxial mesoderm cells in primitive streak at HH stage 4-6 chick embryos.
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