Menzorov, A. G. | Search bioRxiv

bioRxiv2020

Derivation of ringed seal (Phoca hispida) tripotent induced pluripotent stem-like cells

Induced pluripotent stem (iPS) cells have been produced just for a few species among order Carnivora: snow leopard, Bengal tiger, serval, jaguar, cat, dog, ferret, and American mink. We applied the iPS cell derivation protocol to the ringed seal (Phoca hispida) fibroblasts. The resulting cell line had the expression of pluripotency marker gene Rex1. Differentiation in embryoid body-like structures allowed us to register expression of AFP, endoderm marker, and Cdx2, trophectoderm marker, but not neuronal (ectoderm) markers. The cells readily differentiated into adipocytes and osteocytes, mesoderm cell types of origin. Transcriptome analysis allowed us to conclude that the cell line does not resemble human pluripotent cells, and, therefore, most probably are not pluripotent. Thus, we produced ringed seal multipotent stem cell line capable of differentiation into adipo- and osteocytes.

developmental biology↗

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↗