| Unlike most other organs, development of the mammary gland occurs predominantly after birth, under the control of steroid and peptide hormones that are connected to sexual development and reproduction. These defined stages are embryonic, prepubertal, pubertal, pregnancy, lactation and involution. Proliferation and differentiation of secretary mammary epithelium during pregnancy is dependent on the presence of the prolactin receptor (PrlR) and the downstream Jak2-Stat5 pathway. Binding of prolactin or placental lactogens to the PrlR induces receptor dimerization and the phosphorylation of specific tyrosine residues by receptor-associated Jak2. Subsequently, the transcription factors Stat5a and 5b are recruited through their SH2 domains and phosphorylated by Jak2. Phosphorylated Stat5a and Stat5b form homo- or heterodimers and translocate to the nucleus where they activate genetic programs of cell proliferation and differentiation. Stat5a and StatSb belong to Stat family and have a 96% similarity; they exhibit superimposable expression patterns during mammary gland development, although the majority of the differences lie in the transcriptional activation domain at the carboxyl termini. The establishment of mice deficient in StatSa and StatSb has now provided formal proof of this signaling pathway. Stat5a null mice fail to develop functional mammary tissue during pregnancy as a result of reduced epithelium and impaired differentiation. After multiple pregnancies, some functional mammary development can be obtained in StatSa null mice, which coincides with increased StatSb levels, suggesting that StatSb can partially compensate for the absence of StatSa. The deletion of StatSb alone results in a phenotype with similarities to that observed in growth hormone receptor deficient mice. The mice have reduced levels of insulin-like growth factor (IGF-1), reduced growth of males and females. However, those mice that maintained their pregnancy, delivered normal sized litters and were able to lactate. Since Stat5a/b null mice have a nonfunctional corpus luteum and areinfertile, it was not possible to directly assess the combined contribution of both Stat5a and 5b to the development of mammary tissue during the course of a normal pregnancy. Generation of the Stat5a/b conditional gene knockout mouse model may help to understand the true role of Stat5a and Stat5b at any given time point in development when delete the respective genes in defined cell types and during predetermined time windows.To generate the Stat5a/b conditional knockout mouse and fully understand whether other genes in Stat3/5 locus involved in mammary gland development and breast cancer formation, we first obtained 2 BAG clones, which covered 500 kb genomic DNA sequence in this locus, by hybridizing with the mouse BAG library using StatSa, Stat5b and StatB cDNA probes. After sequencing, we searched the NCBI database using the BLAST (nr) algorithm. We found 9 known genes in this locus; they are Ptrf, StatS, Stat5a, Stat5b, Hcrt, BEC2, Gcn5, Cnpl and mDjll, respectively. The strategy of conditional gene knockout was to insert a pair of LoxP at both 3' ends of Stat5a and Stat5b by homologous recombination, when a Cre recombinase transgene is introduced, Cre mediated recombination between the loxP sites can lead to a 90 kb DNA sequence deletion, including the whole genome sequence of Stat5a and Stat5b. Since we are going to delete a huge piece of genomic sequence, it is necessary to know whether the deletion would affect the expression of the neighbor genes in this locus. We analyzed the expression patterns of these 9 known genes in different tissues in mouse. After we delete the Stat5a/b in vivo, we will reanalyze the expressions of these genes to be sure the phenotype is caused by the inactivation of Stat5a/b, not the different expressions of these neighbor genes.We found and cloned two new genes by a combination of bioinformatics and molecular biologic technique, using EST database searches, GENSCAN tools for exon prediction, and cDNA cloning.
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