Chicken β-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish

Abstract

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.

The pro-inflammatory signalling regulator Stat4 promotes vasculogenesis of great vessels derived from endothelial precursors

“…Rescue assays were carried out by co-injection of 50 ng μl −1 mRNA into one-cell-stage embryos. A 4.8 kb nkx2.5 promoter was prepared from Tubigen genome using the forward (5′-GTAGACTGAGTATTCGTTGCGTTAT-3′) and the reverse (5′-TTCCTGACAACAGCCGA - 3′) primers 44 . The N-terminal 51 amino-acid residues truncated stat4 fragment were amplified from cDNA library with the forward primer (5′-CTAGCTAGCATGGCTACTGTACTCTTCAAT-3′) and the reverse primer (5′-CTAGCTAGCGGGTGAACTCATAGCGCTCTC-3′).…”

Activating transcription factor 3 coordinates differentiation of cardiac and hematopoietic progenitors by regulating glucose metabolism

“…We further sought to understand the cell type-specific roles of Atf3 in HPs and CPs. A truncated form of atf3, lacking the C-terminal activation domain, termed atf3 zip , is expressed in the tal1 + HPs and nkx2.5 + CPs under control of the tal1 promoter (17) and the nkx2.5 promoter, respectively (18) (Fig. 4A).…”

EvolvedBmp6enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks

“…Genetic insulators have been reported to limit enhancer activity across the insulator boundary (Bessa et al, 2009;Shimizu & Shimizu, 2013) as well as protect against position effects (Chung et al, 1993), while other experiments show a lack of protection (Grajevskaja et al, 2013). The insulator used here, from the 5'end of the mouse tyrosinase locus, was reported to bind CTCF, like the b-globin 5'HS4 insulator from chicken, and is reported to prevent influences from nearby chromatin state and gene activity, the hallmarks of genetic insulators (Giraldo, 2003;Molto et al, 2009;Montoliu et al, 1996).…”