Alpha-tocopherol transfer protein (ATTP) null mice (ATTP-/-) have a systemic alpha-tocopherol (AT) deficiency, with their lung AT levels being < 10% of those in AT-replete ATTP(+/+) mice when fed a standard rodent chow diet. ATTP(+/+) and ATTP(-/-) mice (4 wk old male mice, n = 16 per group) were fed a standard diet (35 IU AT/kg diet) for 8-12 wk, exposed 6 h/day for 3 days to either to O(3) (0.5 ppm) or filtered air, then sacrificed. No significant differences in plasma or lung AT concentrations were observed in response to this level of O(3) exposure. Lung genomic responses of the lungs to O(3) were determined using Affymetrix 430A 2.0 arrays containing over 22,600 probe sets representing 14,000 well-characterized mouse genes. As compared with filtered air exposure, O(3) exposure resulted in 99 genes being differentially expressed in ATTP(-/-) mice, as compared to 52 differentially expressed genes in ATTP(+/+) mice. The data revealed an O(3)-induced upregulation of genes related to cell proliferation/DNA repair and inflammatory-immune responses in both ATTP(+/+) and ATTP(-/-) mice, with the expression of 22 genes being common to both, whereas 30 and 77 genes were unique to ATTP(+/+) and ATTP(-/-) mice, respectively. The expressions of O(3) sensitive genes-Timp1, Areg, Birc5 and Tnc-were seen to be further modulated by AT status. The present study reveals AT modulation of adaptive response of lung genome to O(3) exposure.
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http://dx.doi.org/10.3109/08958370902838145 | DOI Listing |
BMC Genomics
June 2024
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Kanagawa, Japan.
Background: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci.
View Article and Find Full Text PDFSci Adv
June 2024
University of Colorado School of Medicine, Anschutz Medical Campus, Department of Pediatrics, Section of Developmental Biology, 12801 E 17th Avenue, Aurora, CO 80045, USA.
Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase-based / recombination has streamlined transgenesis in mice and , validated -based landing sites for universal applications are lacking in zebrafish. Here, we developed () as transgenesis approach, with two landing sites and from well-validated Tol2 transgenes.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
February 2022
Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
PH20 is a hyaluronidase enzyme that can hydrolyze the glycosidic bond in hyaluronic acid as the major proteoglycan found in extracellular matrices. In the present study, we constructed and characterized two donor plasmids, one of them with one and the second with two PH20 expression cassettes. The expression vectors were site specifically integrated into the genome of HEK293T cells using PhiC31 integrase system to develop HEK293T stable cell lines secreting His-tagged recombinant human PH20 (rhPH20) in the culture supernatant.
View Article and Find Full Text PDFMethods Mol Biol
December 2020
Applied StemCell, Inc., Milpitas, CA, USA.
Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal and provides an extremely powerful tool to dissect the molecular mechanisms of disease. Transgenic mouse models made by microinjection of DNA into zygotic pronuclei, in particular, have been widely used by the genetics community for over 35 years. However, up till 5 years ago, it remained a rather crude approach: injected sequences randomly insert in multiple copies as concatemers, and they can be mutagenic and have variable, ectopic, or silenced expression depending on the site of integration, a phenomenon called position effects.
View Article and Find Full Text PDFMethods Mol Biol
June 2019
Applied StemCell, Inc., Milpitas, CA, USA.
The discovery of new gene editing tools in the past several years has moved the transgenic field to a new level. The traditional random transgenesis method by pronuclear microinjection has been largely replaced by targeted or site-specific transgenic technologies without the need of homologous recombination in embryonic stem (ES) cells. In this chapter, I describe detailed protocols of an integrase-based approach, trademarked as "TARGATT™" (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into a predetermined chromosomal locus with high efficiency (up to 40%), and faithfully transmitted through generations.
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