Chicken β-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish
- 362 Downloads
Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2 kb cHS4 insulators derived from the chicken β-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site.
KeywordsZebrafish Transposon Position effect Insulator Transgenesis nkx2.5
We thank Dr. Any Wilber for sharing the plasmid containing CHS4 insulators. We thank NIH (Grant HD061749) for financial support.
- Bessa J, Tena JJ, Calle-Mustienes E, Fernández-Miñán A, Naranjo S, Fernández A, Montoliu L, Akalin A, Lenhard B, Casares F, Gómez-Skarmeta JL (2009) Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish. Dev Dyn 238:2409–2417PubMedCrossRefGoogle Scholar
- Clark KJ, Balciunas D, Pogoda HM, Ding Y, Westcot SE, Bedell VM, Greenwood TM, Urban MD, Skuster KJ, Petzold AM, Ni J, Nielsen AL, Patowary A, Scaria V, Sivasubbu S, Xu X, Hammerschmidt M, Ekker SC (2011) In vivo protein trapping produces a functional expression codex of the vertebrate proteome. Nat Methods 8:506–512PubMedCrossRefGoogle Scholar
- Guner-Ataman B, Paffett-Lugassy N, Adams MS, Nevis KR, Jahangiri L, Obregon P, Kikuchi K, Poss KD, Burns CE, Burns CG (2013) Zebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function. Development 140:1353–1363PubMedCrossRefGoogle Scholar
- Ragvin A, Moro E, Fredman D, Navratilova P, Drivenes Ø, Engström PG, Alonso ME, de la Calle Mustienes E, Gómez Skarmeta JL, Tavares MJ, Casares F, Manzanares M, van Heyningen V, Molven A, Njølstad PR, Argenton F, Lenhard B, Becker TS (2010) Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3. Proc Natl Acad Sci USA 107:775–780PubMedCrossRefGoogle Scholar