EFFICIENT TRANSIENT EXPRESSION SYSTEM BASED ON SQUARE PULSE ELECTROPORATION AND INVIVO LUCIFERASE ASSAY OF FERTILIZED FISH EGGS

被引：44

作者：

MULLER, F

LELE, Z

VARADI, L

MENCZEL, L

ORBAN, L

机构：

[1] AGR BIOTECHNOL CTR, INST MOLEC GENET, POB 170, H-2101 GODOLLO, HUNGARY

[2] UNIV AGR SCI, INST ANIM HUSB, GODOLLO, HUNGARY

[3] HUNGARIAN ACAD SCI, INST PLANT PHYSIOL, BIOL RES CTR, H-6701 SZEGED, HUNGARY

来源：

FEBS LETTERS | 1993年 / 324卷 / 01期

关键词：

ELECTROPORATION; GENE TRANSFER; FISH EMBRYO; FIREFLY LUCIFERASE BETA-GALACTOSIDASE; TRANSIENT EXPRESSION;

D O I：

10.1016/0014-5793(93)81525-5

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luceferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

引用

页码：27 / 32

页数：6

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