northern blot membranes  (Mendeley Ltd)

Journal: The Journal of Biological Chemistry

Article Title: The REV7 Subunit of DNA Polymerase ? Is Essential for Primordial Germ Cell Maintenance in the Mouse *

doi: 10.1074/jbc.M112.421966

Figure Lengend Snippet: Establishment of the Rev7-knock-out mouse. A, homologous recombination strategy to generate Rev7−/− mice. The genomic sequence containing Rev7 exon 1 (including the start codon) and exon 2 was replaced by a neomycin cassette. Positions of the 5′ probe for Southern blot screening (gray bars) and PCR primers (arrows) for WT and targeted alleles to screen mouse genotypes are shown. Arrowhead indicates the position of the start codon. B, genotyping of Rev7+/+, Rev7+/−, and Rev7−/− mice by Southern blotting. Genomic DNAs extracted from mouse tails were digested with NheI and Southern hybridization was performed using the 32P-labeled 5′ probe. The 4.66- and 3.96-kb bands represent WT and targeted alleles, respectively. C, genotyping of Rev7+/+, Rev7+/−, and Rev7−/− mice by PCR. The 230- and 750-bp bands represent WT and targeted alleles, respectively. D, Northern blot analysis of total RNA extracted from the testes of Rev7+/+ and Rev7−/− mice. 32P-Labeled full-length Rev7 cDNA was used as the probe (upper panel). Blotting for β-actin expression is shown as the internal control (bottom panel). E, Western blot analysis to detect REV7 protein in tissues of Rev7+/+ and Rev7−/− mice (upper panel). Lysates were prepared from testes, kidneys, liver, and spleen of Rev7+/+ and Rev7−/− mice. Detection of β-actin is shown as the internal control (bottom panel).

Article Snippet: Northern Blot Analysis A Mouse Multiple Tissue Northern blot Membrane was purchased from Clontech.

Techniques: Knock-Out, Homologous Recombination, Sequencing, Southern Blot, Hybridization, Labeling, Northern Blot, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The REV7 Subunit of DNA Polymerase ? Is Essential for Primordial Germ Cell Maintenance in the Mouse *

doi: 10.1074/jbc.M112.421966

Figure Lengend Snippet: Rev7 expression in WT mice. A, Northern blot analysis of Rev7 expression. A Mouse Multiple Tissue Northern blot membrane was hybridized with the 32P-labeled Rev7 cDNA probe (upper panel). A blot hybridized with the β-actin cDNA probe is shown as the internal control (bottom panel). B, Western blot analysis of REV7 expression. Lysates were prepared from P56 WT mice, and Western blotting was performed using the anti-REV7 antibody. REV7 expression was quantitatively assessed using ImageQuant TL, which is indicated at the bottom. C, in situ hybridization to detect the Rev7 transcript in the testis. 33P-Labeled antisense and sense probes for the Rev7 transcript were used for in situ hybridization on testis sections of P56 WT mice. Scale bars, 500 μm. D, immunohistochemical staining for REV7 expression in the testis. Immunostaining was performed with the anti-REV7 antibody on testis sections from P56 WT mice. Positive signals were visualized with DAB or a fluorescently labeled secondary antibody. Immunofluorescence staining without the primary antibody is shown as the negative control (NC). Scale bars, 100 μm. E, in situ hybridization using whole embryonic tissues (upper panels) and tissue sections (bottom panels) to detect Rev7 expression in E9.5 embryos. Whole mount in situ hybridization was performed with DIG-labeled antisense and sense probes for the Rev7 transcript and in situ hybridization on tissue sections was performed with 33P-labeled probes as described under “Experimental Procedures.” In the whole mount in situ hybridization, the purple color throughout the body represents a specific signal (upper left panel). A H&E-stained image of the tissue section is also shown (bottom left panel). Scale bar, 500 μm. F, real-time quantitative RT-PCR analysis of Rev7 expression in E17.5 embryos. Total RNAs were extracted from the indicated organs. Real-time quantitative RT-PCR was performed using gene-specific primers for Rev7 and GAPDH transcripts listed in Table 1. Relative expression of the Rev7 transcript in each organ is graphically shown. Reactions for GAPDH are used as internal controls. G, Western blot analysis of REV7 at postnatal stages. Lysates were prepared from the indicated organs, and Western blot analysis was performed with anti-REV7 and anti-GAPDH antibodies. Relative expression of REV7 was quantitatively assessed using ImageQuant TL, which is indicated at the bottom of each panels. The bands indicated by asterisks are nonspecific.

Article Snippet: Northern Blot Analysis A Mouse Multiple Tissue Northern blot Membrane was purchased from Clontech.

Techniques: Expressing, Northern Blot, Labeling, Western Blot, In Situ Hybridization, Immunohistochemical staining, Staining, Immunostaining, Immunofluorescence, Negative Control, Quantitative RT-PCR