Homologous illegitimate random integration of foreign DNA into the X chromosome of a transgenic mouse line

<p>Abstract</p> <p>Background</p> <p>It is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the <it>fad2 </it>gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration.</p> <p>Results</p> <p>We confirmed that one <it>fad2 </it>line had a sole integration site on the X chromosome according to the inheritance patterns. Mapping of insertion sequences with thermal asymmetric interlaced and conventional PCR revealed that the foreign DNA was inserted into the XC1 region of the X chromosome by a homologous illegitimate replacement of an entire 45,556-bp endogenous genomic region, including the ovarian granulosa cell tumourigenesis-4 allele. For 5' and 3' junction sequences, there were very short (3-7 bp) common sequences in the AT-rich domains, which may mediate the recognition of the homologous arms between the transgene and the host genome. In addition, analysis of gene transcription indicated that the transgene was expressed in all tested <it>fad2 </it>tissues and that its transcription level in homozygous female tissues was about twice as high as in the heterozygous female (p < 0.05).</p> <p>Conclusions</p> <p>Taken together, the results indicated that the foreign <it>fad2 </it>behaved like an X-linked gene and that foreign DNA molecules were inserted into the eukaryotic genome through a homologous illegitimate random integration.</p

Molecular Mechanisms of Bladder Outlet Obstruction in Transgenic Male Mice Overexpressing Aromatase (Cyp19a1)

We investigated the etiology and molecular mechanisms of bladder outlet obstruction (BOO). Transgenic (Tg) male mice overexpressing aromatase (Cyp19a1) under the ubiquitin C promoter in the estrogen-susceptible C57Bl/6J genetic background (AROM+/6J) developed inguinal hernia by 2 months and severe BOO by 9 to 10 months, with 100% penetrance. These mice gradually developed uremia, renal failure, renal retention, and finally died. The BOO bladders were threefold larger than in age-matched wild-type (WT) males and were filled with urine on necropsy. Hypotrophic smooth muscle cells formed the thin detrusor urinae muscle, and collagen III accumulation contributed to the reduced compliance of the bladder. p-AKT and ERα expression were up-regulated and Pten expression was down-regulated in the BOO bladder urothelium. Expression of only ERα in the intradetrusor fibroblasts suggests a specific role of this estrogen receptor form in urothelial proliferation. Inactivation of Pten, which in turn activated the p-AKT pathway, was strictly related to the activation of the ERα pathway in the BOO bladders. Human relevance for these findings was provided by increased expression of p-AKT, PCNA, and ERα and decreased expression of PTEN in severe human BOO samples, compared with subnormal to mild samples. These findings clarify the involvement of estrogen excess and/or imbalance of the androgen/estrogen ratio in the molecular pathogenetic mechanisms of BOO and provide a novel lead into potential treatment strategies for BOO

The Potential of Rat Inner Cell Mass and Fetal Neural Stem Cells to Generate Chimeras

The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses (ICMs) and fetal neural stem (FNS) cells. In result, three rat chimeras were produced by day 5 (D5) Sprague-Dawley (SD) blastocysts injected with ICMs derived from day 6 (D6) and D5 Dark Agouti (DA) blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs are able to contribute to chimeras, but their potential decreases significantly after culture in vitro (P<0.05), and rFNS cells only have the potential to contribute to early fetal development