Expression of the 11β-hydroxysteroid dehydrogenase 2 gene in the mouse

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Expression of the 11β-hydroxysteroid dehydrogenase 2 gene in the mouse Xiao-Lei Moore, Isabelle Hoong, Dr Timothy J. Cole Kidney International Volume 57, Issue 4, Pages 1307-1312 (April 2000) DOI: 10.1046/j.1523-1755.2000.00967.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 DNA sequence and transcription start-site mapping of the mouse HSD2 gene promoter. (A) DNA sequence of 500 nucleotides upstream of the mouse HSD2 gene (M) and sequence comparison with the human HSD2 gene (H), including part of exon 1. The ATG translation start in exon 1 is boxed. Binding sites in the human HSD2 sequence for the SP1 transcription factor are boxed. Transcription start sites in both the mouse (B) and human (18) HSD2 sequences are indicated by stars. (B) Mapping of transcription start sites for the mouse HSD2 gene. Total RNA (15 μg) from liver, kidney, and colon (in duplicate) was incubated with a 460 nucleotide Sma I-Pst I HSD2 antisense riboprobe (Probe) from the proximal promoter region, including part of exon 1. Yeast tRNA (tRNA) was incubated as a negative control. Protected fragments of approximately 180 nucleotides were detected for kidney and colon only. Kidney International 2000 57, 1307-1312DOI: (10.1046/j.1523-1755.2000.00967.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 DNA sequence and transcription start-site mapping of the mouse HSD2 gene promoter. (A) DNA sequence of 500 nucleotides upstream of the mouse HSD2 gene (M) and sequence comparison with the human HSD2 gene (H), including part of exon 1. The ATG translation start in exon 1 is boxed. Binding sites in the human HSD2 sequence for the SP1 transcription factor are boxed. Transcription start sites in both the mouse (B) and human (18) HSD2 sequences are indicated by stars. (B) Mapping of transcription start sites for the mouse HSD2 gene. Total RNA (15 μg) from liver, kidney, and colon (in duplicate) was incubated with a 460 nucleotide Sma I-Pst I HSD2 antisense riboprobe (Probe) from the proximal promoter region, including part of exon 1. Yeast tRNA (tRNA) was incubated as a negative control. Protected fragments of approximately 180 nucleotides were detected for kidney and colon only. Kidney International 2000 57, 1307-1312DOI: (10.1046/j.1523-1755.2000.00967.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 Tissue expression profile of mouse HSD1 and HSD2 by RNase protection assay. Expression of HSD1 and HSD2 mRNA was detected by RNase protection in total RNA using 32P-labeled antisense probes for HSD1, a 270-bp Xba I cDNA fragment encompassing exons 4 through 6, HSD2, and a 320 bp Sty I-EcoRI fragment from exon 5. Total RNA (10 μg) from various tissues was incubated with both probes (Probe) together overnight at 56°C. Yeast tRNA (tRNA) was incubated as a negative control, and size marker (M) was end labeled 1 kb ladder (GIBCO BRL). Kidney International 2000 57, 1307-1312DOI: (10.1046/j.1523-1755.2000.00967.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Targeting strategy for tissue-specific gene targeting of the mouse HSD2 gene. The neomycin resistance gene (neor), the thymidine kinase gene (TK), and three 34 bp lox P binding sites (arrows) were inserted into the mouse HSD2 gene (mHSD2) to produce a conditional targeting vector for introduction into mouse ES cells. Probe A (0.7 kb BglII-PvuII fragment) and probe B (1.4 kb Xba I fragment) are single copy probes for Southern analysis of Eco RI/BamHI and Hind III enzyme digests of genomic DNA, respectively. Exons 1 through 5 of the HSD2 gene are shown as filled boxes. Kidney International 2000 57, 1307-1312DOI: (10.1046/j.1523-1755.2000.00967.x) Copyright © 2000 International Society of Nephrology Terms and Conditions