Genetic manipulation of mammalian dictyate oocytes: factors affecting transient expression of microinjected DNA templates.
Transcription of exogenous DNA templates in mouse ovarian oocytes was investigated by microinjecting constructs encoding for the Escherichia coli lacZ gene under control of promoters from: 1) the mouse hsp68 gene; 2) the human beta-actin gene; and 3) simian virus 40 (SV40) early genes. Various amounts of circular or linear DNA constructs were injected into dictyate oocyte nuclei at different stages of follicle growth, and the beta-galactosidase activity was then cytochemically evaluated in single cells. In middle-sized growing oocytes, expression of circular constructs was observed with amounts of DNA ranging from 50 to 10(3) plasmid copies/nucleus and was first observed 10-12 hr after injection. Maximal expression levels were reached by 17 hr after injection and were specific for the constructs used. Circular constructs containing the hsp68 and early SV40 promoters were expressed at similar levels in small- and middle-sized growing oocytes, while the construct carrying the beta-actin promoter was expressed only in small-sized cells. In contrast to growing oocytes, these constructs were never expressed in fully grown oocytes. DNA linearization depressed construct activity regardless of the site of cleavage. These results show that: 1) lacZ is a valuable reporter gene in the analysis of eukaryotic promoter activity in dictyate mouse oocytes; 2) transient construct expression requires the injection of DNA in circular form; and 3) the expression efficiency of different DNA templates is dependent on the presence of a specific promoter and on the differentiation stage of oocytes analyzed.
Bevilacqua
A
A
Department of Psychology, University La Sapienza, Rome, Italy.
Kinnunen
L H
LH
Mangia
F
F
D000199:Actins / Q000235:genetics; D000818:Animals; D004270:DNA, Circular; D004274:DNA, Recombinant / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011418:Prophase; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013539:Simian virus 40 / Q000235:genetics; D013698:Templates, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D000199:Actins; D004270:DNA, Circular; D004274:DNA, Recombinant; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase