A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice

Abstract

A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.

BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene

“…2), which is known to be regulated by an enhancer located 20 kb upstream of the SNP (Ragvin et al ., 2010). This is no proof, but from our enhancer detection screen and another transposon‐based enhancer detection approach we find that enhancer activity decreases with increasing distance from the gene (Kikuta et al ., 2007b; Kokubu et al ., 2009). Other support for this notion comes from p300 and H3K27ac ChIP sequencing data produced in embryonic and fetal mouse forebrain (Visel et al ., 2013).…”

Sleeping Beauty Transposition

“…For example, the P-element transposon of Drosophila prefers to insert within ∼100 kb of the donor site at a rate ∼50-fold higher than in regions outside that interval (132). Similarly, 30 to 80% of SB transposition events were found to re-insert locally on either side of the transposon donor locus (93,133,134,135,136,137,138,139). In contrast to the P-element, SB seems to have a much larger local transposition interval (in the megabase range), but the targeted window for local reinsertion appears to be dependent on the donor locus and can range from <4 Mb (140) to 5 to 15 Mb (134).…”

Germline transgenesis of the chordate Ciona intestinalis with hyperactive variants of sleeping beauty transposable element

“…If an SB transgenic line that has an insertion close to a gene-of-interest is isolated, remobilization of the insertion will result in a higher probability of isolating mutant lines that have insertions into the targeted gene compared with random mutagenesis. Finally, deletion of the C. intestinalis genome can be done by using intrachromosomal transposition of SB and Cre-loxP systems, as was shown in mouse (Kokubu et al, 2009). For this purpose, the combinatorial use of SB and Minos is essential.…”