T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied by polyethylenimine based in vivo gene transfer.
The formulation of cationic polymers of polyethylenimine (PEI) with plasmid DNA has been optimized to deliver genes into the Xenopus tadpole brain in vivo. Using intraventricular microinjections of 1 microl (containing 0.5 to 1 microg DNA) we show that the linear, low molecular weight polymer, 22 kDa PEI was significantly more efficient than a branched 25 kDa polymer. Complexes bearing a slightly positive net charge (formed with a ratio of 6 PEI amines per DNA phosphate) provided the best levels of transfection. Transgene expression was DNA-dose dependent and was maintained over 6 days, the time course of the experiment. Spatial distribution was examined using a beta-galactosidase construct and neurones expressing this transgene were found spread throughout the brain. The possibility of using this technique to evaluate physiological regulations was approached by examining the effects of tri-iodothyronine (T3), on transcription from the mammalian TRH and Krox-24 promoter sequences. Adding physiological concentrations of T3 to the aquarium water significantly reduced transcription from the rat TRH promoter whilst the same treatment increased transcription from a mouse Krox-24 -luciferase construct. Thus, PEI-DNA transfection provides a versatile and easily applied method for following physiological regulations at the transcriptional level in the tadpole brain.
Ouatas
T
T
Laboratoire de Physiologie Générale et Comparée, Muséum National d'Histoire Naturelle, URA 90 CNRS, Paris, France.
Le Mével
S
S
Demeneix
B A
BA
de Luze
A
A
D000818:Animals; D001921:Brain / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D051766:Early Growth Response Protein 1; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017874:Immediate-Early Proteins; D007700:Kinetics; D007814:Larva / Q000378:metabolism; D008970:Molecular Weight; D011094:Polyethyleneimine; D011401:Promoter Regions, Genetic; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D014284:Triiodothyronine / Q000494:pharmacology*; D014981:Xenopus / Q000254:growth & development* / Q000378:metabolism*; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; C486111:EGR1 protein, Xenopus; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; D014284:Triiodothyronine; D013973:Thyrotropin-Releasing Hormone; D011094:Polyethyleneimine