Effect of CaCl2 concentration on the rate of foreign gene transfer and expression by in vivo electroporation in the mouse ovary.
We tested the effect of electroporation (EP) medium composition on the rate of gene transfer and expression in the mouse ovary in vivo. FITC labeled oligonucleotides were dissolved in a medium with varying levels of CaCl2 concentration from 0 to 250 mM, and transferred by in vivo EP. Gene transfer efficiency was assessed by examining fluorescence signal intensity with a fluorescent microscope at 3 h after in vivo EP. The results indicated that CaCl2 concentration at 50 mM gave the highest transfer efficiency of the oligonucleotides only in the presence of phosphate buffered saline (PBS). Without PBS or CaCl2, the oligonucleotide transfer was negligible. A further increase in CaCl2 from 50 to 250 mM lowered the transfer efficiency. Little fluorescence signal was attained by substituting CaCl2 for MgCl2, NaCl or KCl. Addition of glycerol to the EP medium with 50 mM CaCl2 did not improve the transfer efficiency in the presence of PBS, although a marginal increase was observed in the absence of PBS. The stimulating effect of increased CaCl2 concentration from 0 to 50 mM was further evaluated by examining the intensity of reporter protein expression after transferring the bacterial lacZ gene. The results of X-gal staining demonstrated that CaCl2 with a range of 20 to 100 mM, showed enhanced gene expression in comparison with 0 mM. However, no remarkable difference was observed between the different CaCl2 concentrations, suggesting that the stimulating effect of CaCl2 on gene transfer and expression in the mouse ovary in vivo may not necessarily parallel in terms of the optimal concentration.
Suzuki
Takayuki
T
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Tsunekawa
Jun
J
Murai
Atsushi
A
Muramatsu
Tatsuo
T
D000818:Animals; D002122:Calcium Chloride / Q000378:metabolism*; D003470:Culture Media; D004247:DNA / Q000378:metabolism*; D018274:Electroporation; D005260:Female; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005990:Glycerol; D051379:Mice
D003470:Culture Media; D004247:DNA; D002122:Calcium Chloride; D005990:Glycerol