Generation of transgenic newt Cynops pyrrhogaster for regeneration study.
To take advantage of the ample potential for tissue regeneration by the newt, a technique to create transgenic newt was developed. The technique was based on a procedure for producing transgenic Xenopus, but modified to adapt to the different sperm morphology and to overcome the refractoriness of newt eggs to activation by normal cleavage. Sperm was collected from mature testes early in winter, permeabilized with digitonin, but without treatment of egg extract. Efficient egg activation was achieved by coinjection of inositol 1,4,5-trisphosphate (IP3) with DNA-sperm nucleus complex. Transgenic Cynops for EGFP/DsRed2 genes under the control of cytomegalovirus (CMV) enhancer/promoter showed nonmosaic widespread expression of reporter genes in embryos, swimming larvae, and adults after metamorphosis. Transgenic newt carrying EGFP gene under regulation of betaB1-crystallin promoter expressed the transgene uniquely in the lens. During lens regeneration after lens removal, EGFP expression occurred, reflecting the lens regeneration process. The newt transgenesis technique described here is likely to be of wide use in monitoring and manipulating gene expression in the study of molecular mechanisms underlying tissue regeneration.
Ueda
Yoko
Y
Differentiation Transition Group, Kondoh Differentiation Signaling Project, ERATO, Japan Science and TechnologyCorporation, and Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
Kondoh
Hisato
H
Mizuno
Nobuhiko
N
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D007908:Lens, Crystalline / Q000502:physiology; D008297:Male; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012038:Regeneration / Q000235:genetics* / Q000502:physiology; D012455:Salamandridae / Q000235:genetics* / Q000502:physiology*; D038206:beta-Crystallin B Chain / Q000235:genetics
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D038206:beta-Crystallin B Chain; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins