Biology subjects

Fishman, V.

Publications and source records attributed to Fishman, V..

3 recordsLinked to original sources

bioRxiv2019

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

Mechanisms that ensure repair of double-stranded DNA breaks play a key role in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found in the form of concatemer consisting of multiple co-integrated transgene copies. Here we investigated contribution of various DSB repair pathways to the concatemer formation. We injected a pool of linear DNA molecules carrying unique barcodes at both ends into mouse zygotes and obtained 10 transgenic embryos with transgene copy number ranging from 1 to 300 copies. Sequencing of the barcodes allowed us to assign relative positions to the copies in concatemers and to detect recombination events that happened during integration. Cumulative analysis of approximately 1000 integrated copies revealed that more than 80% of copies underwent recombination when their linear ends were processed by SDSA or DSBR. We also observed evidence of double Holliday junction (dHJ) formation and crossing-over during the formation of concatemers. Additionally, sequencing of indels between copies showed that at least 10% of the DNA molecules introduced into the zygote are ligated by non-homologous end joining (NHEJ). Our barcoding approach documents high activity of homologous recombination after exogenous DNA injection in mouse zygote.

molecular biology↗

bioRxiv2019

Quantitative prediction of enhancer-promoter interactions

Recent experimental and computational efforts provided large datasets describing 3-dimensional organization of mouse and human genomes and showed interconnection between expression profile, epigenetic status and spatial interactions of loci. These interconnections were utilized to infer spatial organization of chromatin, including enhancer-promoter contacts, from 1-dimensional epigenetic marks. Here we showed that predictive power of some of these algorithms is overestimated due to peculiar properties of biological data. We proposed an alternative approach, which gives high-quality predictions of chromatin interactions using only information about gene expression and CTCF-binding. Using multiple metrics, we confirmed that our algorithm could efficiently predict 3-dimensional architecture of normal and rearranged genomes.

genomics↗

bioRxiv2018

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: EGFP under pan-neural marker class III {beta}-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP ES cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on mRNA level using PCR-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared to random genomic integration.

developmental biology↗