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Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.
The bovine adrenodoxin gene gives rise to two species of mRNA differing only at their 5'-ends. The synthesis of these two types of mRNA in bovine adrenal cortical cells is regulated transcriptionally in part by ACTH via the action of cAMP. Examination of the 5'-end of the adrenodoxin gene revealed that each mRNA contains sequences derived from a different exon encoding the mitochondrial leader sequence. To define the sequences necessary for the synthesis of these two types of mRNA and to determine if the synthesis of each is driven by a separate promoter, 5'-regions of the adrenodoxin gene were inserted upstream from two different reporter genes and tested for promoter/enhancer regulatory activity by transient transfection into mouse adrenocortical Y1 tumor cells. The results clearly demonstrated that the bovine adrenodoxin gene contains two functional promoters; one, ADXP1, located in the 5'-flanking region gives rise to the minor form of mRNA, and a second, stronger promoter, ADXP2, which maps within intron 1 gives rise to the major form of mRNA. Unique cAMP-responsive sequences were found upstream from each promoter which share no sequence homology to the consensus CRE (cAMP-responsive element). Upon transient expression, the cAMP-responsive sequence associated with the ADXP2 promoter, termed CRS2, confers the cAMP responsiveness to stimulate the transcription of the linked beta-globin reporter gene regardless of whether the adrenodoxin ADXP2 promoter or the beta-globin promoter was utilized.(ABSTRACT TRUNCATED AT 250 WORDS)
We describe transgenic mouse lines that express lacZ under the control of the Hox 3.3 Promoter II. The correct anterior boundary can be fixed by 3.6 kb of promoter DNA (plus 1.6 kb of 5' transcribed sequences), both in tissues of ectodermal and mesodermal origin. The posterior border, however, is not respected, and lacZ expression continues into the tail region. One line has particularly strong graded expression in the anterior proximal limb bud. Other lines, containing a shorter promoter fragment (0.6 kb), have ectopic expression in the head region, including one line that has expression in the anterior half of the retina. Such mouse lines make it possible to molecularly distinguish cells in regions of the embryo that look otherwise identical and may be useful in studying the establishment of molecular differences in the mouse embryo.