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Investigating L1 promoter activity during mouse development using LacZ transgenes
Author(s) -
Saha Partha S.,
LaCanne Trenton,
An Wenfeng
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00748
Subject(s) - transgene , biology , reporter gene , gene , promoter , dna methylation , genetics , epigenetics , lac operon , retrotransposon , locus (genetics) , microbiology and biotechnology , gene expression , genome , transposable element
Long interspersed elements type 1 (L1s) are highly abundant retrotransposons in the human genome. To fully understand the role of L1s during development and disease, it is important to track L1 expression and epigenetic regulation at individual L1 loci throughout development at the cellular level. However, it is extremely technically challenging to monitor transcriptional activities of individual endogenous L1s due to high sequence homology. Toward this goal, we created independent single‐copy LacZ transgenic mouse lines, in which the LacZ reporter is regulated by a copy of the endogenous mouse L1 promoter. To control for the effect of gene‐body DNA methylation on L1 promoter activity, we also generated independent mouse lines carrying the CpG‐less version of the LacZ, termed LacG. After mapping of the transgene locus in each line, mouse tissues were stained with X‐gal to visualize L1 promoter activity at single cell level. QuPath was used to quantify X‐gal positive cells in whole‐slide scanned brain sections. We found that the LacG reporter supported robust expression as compared to the LacZ reporter. However, there were substantial variations among the LacG reporter lines, in which the transgene is integrated in random genomic loci. Intraline variations of expression in the same organ might be dictated by generation and age, which is yet to be established statistically. Orientation dependent regulation of L1 promoter was also studied by targeting the LacG transgene in a specific intronic locus in either sense or antisense orientation. Interestingly, antisense orientation showed augmented expression than its counterpart. Ongoing effort aims to reveal the pattern of these signals in different anatomical regions of brain and also in other major organs, which might support L1 retrotransposition in different developmental period in these mouse models. The goal is to provide a comprehensive mapping of L1 promoter activity in a cell dependent and locus dependent manner. Such information will be instrumental in our understanding of L1’s involvement in physiological and pathological conditions, including embryogenesis, gametogenesis, and under environmental stress. Support or Funding Information Supported by NIH R21HD080143 and R21OD017965.