Expression Of BRD7in Mouse Tissues And Construction Of BRD7Knock-out Mice

BRD7which is cloned by our lab using cDNA representational differential assay acts as a candidate tumor suppressor gene to prevent cell cycle Gl-S phase progression, initiating apoptosis, reversing cell malignant. BRD7is one of bromodomain family members that can bind and recognize of acetylated histone H3, and get involved in ras/MEK/ERK, Rb/E2F and Wnt signaling pathways, showing its function in tumor inhibition. Brd7is a nuclear transcription regulator that control nasopharyngeal cancer cell cycle and apoptosis.Although the research of BRD7’s function and mechanism in tumor inhibition has made breakthrough progress, we still need experimental evidence in vivo. Recently, gene knockout technology can use homologous recombination to inactivated target genes, and then analyze the gene’s function with its lacked. By knockout, a series of phenotypic changes can be reflected in the whole physical and cell level. Here we use C57BL/6mice as a research model, to compare murine and human BRD7gene in the mRNA sequence and the amino acid sequence homology and to study the expression of BRD7in all kinds of murine tissues, on the other hand, using Cre/LoxP system to explore the construction of knockout mice model and identification, construct the BRD7conditional knockout mouse models. This research is aimed to find tumor suppressive function of BRD7in vivo and lay a solid foundation for comprehensive studying of BRD7physiologically and pathologically. The experimental results are as followed: The murine BRD7’s sequence characteristics and expression1. Sequence characteristics of a murine BRD7C57BL mice are a commonly used gene knockout animal models.Its coding frame sequence of Brd7is1956bp, containing17exons, encoding651amino acids. While coding frame sequence of BRD7in human is1956bp, containing13exons also encoded651amino acids. Homology between mouse and human in mRNA and amino acid sequence is up to88%, indicating that the functions of Brd7in C57BL mice is almost the same as human. For that reason, C57BL is the best type of mice for the construction of BRD7knockout mice.2. The expression pattern of mBRD7in each tissue of miceDigital expression analysis （electronic hybrid） of mBrd7showing a higher expression level in the brain, eyes, heart, liver, lung, pancreas, skin, testes, intestines, blood, bone and prostate but lower expression in the muscle, salivary gland, stomach. This results show that Brd7is a conservative gene which is widely expressed. Fluorescence quantitative PCR found that the average CT value of mBRD7is24.2cycle with GAPDH is19.0cycle, the mRNA expression levels of BRD7is1/36.76of GAPDH. The expression of BRD7in the middle/low level in mouse tissue, but higher expression in male reproductive organization as testis, epididymis, seminal vesicles, seminiferous tubes, as well as the spleen, lung, ear and other organizations, suggesting that BRD7may be somewhat association with the male mouse fertility.The construction of BRD7knockout mice:1. The construction and identification of BRD7LoxP1-2+/-miceThe insertion of LoxP is the most important step in conditional construction of knockout mice, which is cooperated with Shanghai south model organism research centre. The preparation process including the construction of targeted vector, transduction of ES cells, injection of recombination ESC, selection for positive clone. We select BRD7-LoxP1₂+/-through genotype identification and fur color. gDNA of each mice is amplified by LoxPl,LoxP2and WT primer to prove that BRD7-LoxP1-2+/-is successful constructed.2. The construction and identification of BRD7LoxP3+/+miceMate the male and female mice of BRD7LoxP1-2+/-, using primers such as LoxP1, LoxP2and WT to by PCR with their gDNA, in this way we can obtain the genotype of BRD7LoxP1₂+/+mice; then mate with EⅡ Cre+/+mice. Using Primes of LoxP1, LoxP2, LoxP3, WT, and Cre to do PCR to select the genotype of BRD7LoxP3+/-Cre+/-mice; then hybridized with BRD7loxP1-2+/+Cre-/-mice to obtain the genotype of BRD7loxP3+/+, which is BRD7-/-knockout mice in theoretic. 3. The identification of BRD7gene knockout （KO） miceOn the basis of successfully construct BRD7LoxP3+/+（BRD7-/-） mice, we do PCR by PLoxP3primers to amplificate LoxP3clips, then do plastic recycling to purified fragment and sequencing, in that way we confirmed the exon3-4of BRD7is successfully removed from gDNA level; use the BRD7LoxP1-2+/+and BRD7LoxP3+/+mice as the study model, extract the total RNA of colorectal and do reverse transcription experiment, do PCR and sequencing with the primers of BRD7overall Length and specific primers for the exon3-4to confirmed BRD7gene knockout mice were successfully constructed from the size of mRNA fragment and the characteristics of sequence; finally, we randomly selected two of wild-type mice and knockout mice, extract spleen RNA and do reverse transcription experiment to confirmed BRD7gene knockout mice were successfully constructed from the mRNA and organizational level.4. The mass-production of BRD7knockout miceIn order to avoid the impact of Cre itself on mouse, we select BRD71oxP3+/LoxP1-2EⅡ-Cre-/-mice mate with the same genotype, so the mass-production of BRD7knockout mice is available. Now there are36knockout mice,82BRD7+/-mice and90BRD7+/+have been identified, These mice lay a solid foundation for us to fully explore the phenotype and molecular mechanisms of BRD7in mice. In summary, this study found that the mice BRD7in C57BL and the human BRD7are highly homology, both of them encoding651amino acids, widely expressed in the mouse tissues, but higher expression have been found in male reproductive-related organizations like testis, epididymis, seminal vesicles, as well as other organizations like spleen, lung, ea.. In this study, we put C57BL as a mouse model, use Cre/LoxP system to construct Brd7knockout mice, and in order to explore a effective method in construction and identification for knockout mice. We also reproduce a large number of BRD7knockout mice, heterozygote and wild type mice, laid solid foundation for comprehensive study the process and mechanism of BRD7how to participate in normal physiology and disease in vivo.