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- Cederberg, Anna, 1972, et al.
(författare)
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In vitro differentiated adipocytes from a Foxc2 reporter knock-in mouse as screening tool.
- 2009
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Ingår i: Transgenic research. - : Springer Science and Business Media LLC. - 1573-9368 .- 0962-8819. ; 18:6, s. 889-97
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a generic model for in vitro high-throughput screening for agents regulating transcription of genes in the mouse genome here exemplified by Foxc2, a forkhead transcription factor involved in regulation of adipocyte metabolism. We made a Foxc2-LacZ reporter "knock-in" mouse in which one of the two Foxc2 alleles has been inactivated and replaced by a LacZ reporter gene. Mouse embryonic fibroblasts, derived from such mice, were differentiated in vitro to adipocytes and used in cell-based screens. Forskolin as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) increased levels of Foxc2nLacZ fusion protein. We could also demonstrate that this was paralleled by an increase in Foxc2 mRNA, transcribed from the wild type allele. This generic method offers a novel way of identifying both positive and negative upstream regulators of a gene, using high-throughput screening methodology. In a cell-based screen using such methodology we demonstrate efficacy by identifying NKH477 as a Foxc2 activating compound.
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| 3. |
- Wadenback, Johan, et al.
(författare)
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Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR)
- 2008
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Ingår i: Transgenic research. - : Springer Science and Business Media LLC. - 0962-8819 .- 1573-9368. ; 17:3, s. 379-392
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Tidskriftsartikel (refereegranskat)abstract
- An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.
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- Yang, Shao H., et al.
(författare)
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Caution! Analyze transcripts from conditional knockout alleles.
- 2009
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Ingår i: Transgenic research. - : Springer Science and Business Media LLC. - 1573-9368 .- 0962-8819. ; 18:3, s. 483-9
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Tidskriftsartikel (refereegranskat)abstract
- A common strategy for conditional knockout alleles is to "flox" (flank with loxP sites) a 5' exon within the target gene. Typically, the floxed exon does not contain a unit number of codons so that the Cre-mediated recombination event yields a frameshift and a null allele. Documenting recombination within the genomic DNA is often regarded as sufficient proof of a frameshift, and the analysis of transcripts is neglected. We evaluated a previously reported conditional knockout allele for the beta-subunit of protein farnesyltransferase. The recombination event in that allele-the excision of exon 3-was predicted to yield a frameshift. However, following the excision of exon 3, exon 4 was skipped by the mRNA splicing machinery, and the predominant transcript from the mutant allele lacked exon 3 and exon 4 sequences. The "Deltaexon 3-4 transcript" does not contain a frameshift but rather is predicted to encode a protein with a short in-frame deletion. This represents a significant concern when studying an enzyme, since an enzyme with partial function could lead to erroneous conclusions. With thousands of new conditional knockout alleles under construction within mouse mutagenesis consortiums, the protein farnesyltransferase allele holds an important lesson-to characterize knockout alleles at both the DNA and RNA levels.
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