LEXSY Transfection and Selection Protocols - Illustrative overview -

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1 LEXSY Transfection and Selection Protocols - Illustrative overview - Jena Bioscience GmbH Loebstedter Str Jena, Germany Tel.: Fax: info@jenabioscience.com

2 Alternative LEXSY electroporation protocols - to meet the equipment available in your lab - Electroporation in LEXSY BHI ca. 0 8 cells/ml Low voltage protocol High voltage protocol Exptl. decay d=mm V=0. ml Time constant d=mm V=0. ml d=mm V=0. ml set: 0V set: 0V 0 µf T=.ms pulse pulse set: 00V µf 0 interval get: - 6 ms 0 Ω 0 V deliv. get:. ms 0 Ω 0 V deliv. get:. ms 0 Ω 00 V delivered Present standard method at Jena Bioscience ) Beverley SM, Clayton CE (99) Methods in Molecular Biology, Vol.: Protocols of Molecular Parasitology chapter p. -7 ) Robinson KA & Beverley SM (00) Mol. Biochem. Parasitol. 8: 7-8

3 Low voltage protocol for transfection of Leishmania tarentolae grow suspension culture at 6 C in LEXSY BHI medium to ca. 6x0 7 cells/ml (OD ca..) concentrate to 0 8 cells/ml by centrifugation min/000g at RT and removal of ½ of SN place cells on wet ice for 0 min add 0. ml of cold cells to - 0 µg DNA in 0 µl water/buffer kept on ice transfer to prechilled electroporation cuvette d=mm pulse at 0 V, 0 µf; monitor T ( - 6 ms), exponential decay ) or at 0 V,. ms; monitor T (. ms), time constant protocol put cuvettes back on ice for EXACTLY 0 min transfer to 0 ml LEXSY BHI medium kept at 6 C incubate o/n at 6 C (6- h) add selection antibiotic(s) to suspension culture for selection of cell lines (chapt.. of constitutive LEXSY manual) and/or spin min/000g, resuspend to ca. 0. ml and plate on LEXSY BHI agar + selective drug(s) for clonal selection (chapt... of constitutive LEXSY manual) References: ) Beverley SM, Clayton CE (99) Methods in Molecular Biology, Vol.: Protocols of Molecular Parasitology chapter p. -. Detailed description in LEXSY manuals capt../.:

4 High voltage protocol for transfection of Leishmania tarentolae grow suspension culture at 6 C in LEXSY BHI medium to ca. 6x0 7 cells/ml (OD ca..) concentrate to 0 8 cells/ml by centrifugation min/000g at RT and removal of ½ of SN place cells on wet ice for 0 min add 0. ml of cold cells to - 0 µg DNA in 0 µl water/buffer kept on ice transfer to prechilled electroporation cuvette d=mm pulse times at 00 V, µf (0 sec between pulses), monitor T (. ms) ) put cuvettes back on ice for exactly 0 min transfer to 0 ml LEXSY BHI medium kept at 6 C incubate o/n at 6 C (6- h) add selection antibiotic(s) to suspension culture for selection of cell lines (capt.. of LEXSY manual) and/or spin min/000g, resuspend to ca. 0. ml and plate on LEXSY BHI agar + selective drug(s) for clonal selection (capt... of LEXSY manual) References: ) Robinson KA, Beverley SM. (00) Mol Biochem Parasitol. 00 8():7-8. Detailled description in LEXSY manuals capt. 8..

5 Selection of recombinant Leishmania lines in suspension culture Transfection (expression plasmid) living cells :0 living cells Expansion of recombinant suspension culture by :0 dilution -7 d drug turbid -7 d drug turbid Confirmation of genetic structure Evaluation of target protein expression Strain conservation living & dead cells :0 few dead cells Optimization of expression profile Scaling up... Electroporation 0 V 0 µf. msec d=mm V=0. ml -7 d drug turbid -7 d drug Negative control (no DNA) Detailled description in LEXSY manuals capt..: clear

6 Microscopic control of LEXSY selection procedure Negative control (no DNA) with selection dead cells Recombinant culture with selection healthy motile cells

7 Clonal selection of expression clones on BHI agar Negative control (no DNA) expression clones - pick clones at >- mm colony diameter ca. 7-9 days after plating - expand in ml BHI medium in well 0 ml formats at 6 C under selection - confirm genetic structure, evaluate target protein expression - prepare glycerol stocks Detailed description in LEXSY manuals capt. and appendix 8.: 6

8 Determination of MIC for LEXSY selection For optimal selection efficiency the minimal inhibitory concentrations (MIC) of selective antibiotics should be determined. Use of JBS LEXSY antibiotics circumvents the need for time consuming MIC determination for drugs from other suppliers. For optimal results use LEXSY NTC, LEXSY Bleo and LEXSY Hygro at 00 µg/ml, and LEXSY Neo at 0 µg/ml. MIC LEXSY NTC MIC LEXSY Bleo Growth OD 78 nm 0 ug/ml ug/ml 0 ug/ml 00 ug/ml 00 ug/ml Growth OD 78 nm 0 ug/ml ug/ml 0 ug/ml 00 ug/ml 00 ug/ml Time (hours) Time (hours) MIC LEXSY Hygro MIC LEXSY Neo Growth OD 78 nm Series 0 ug/ml ug/ml 0 ug/ml 00 ug/ml 00 ug/ml Growth OD 78 nm 0 ug/ml ug/ml 0 ug/ml 0 ug/ml Time (hours) MIC: minimal inhibitory concentration Time (hours) 7