Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check.

Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice.

Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals.

Mind bomb 1 is required for pancreatic β-cell formation.

During early pancreatic development, Notch signaling represses differentiation of endocrine cells and promotes proliferation of Nkx6-1(+)Ptf1a(+) multipotent progenitor cells (MPCs). Later, antagonistic interactions between Nkx6 transcription factors and Ptf1a function to segregate MPCs into distal Nkx6-1(-)Ptf1a(+) acinar progenitors and proximal Nkx6-1(+)Ptf1a(-) duct and β-cell progenitors. Distal cells are initially multipotent, but evolve into unipotent, acinar cell progenitors.

Identification of a conserved cis-acting region driving expression of mouse Eomesodermin to the primitive streak, node, and definitive endoderm.

The cis-acting elements that regulate Eomes transcription during embryonic development are largely unknown. Here we identify a conserved cis-acting region (EoIV) located ~20kb upstream of the Eomes coding region that faithfully drives reporter expression to sites of Eomes expression during gastrulation. Transgenic EoIV-hsp68-GFP expression was evident in the epiblast of early-streak stage mouse embryos at the site where the primitive streak is initiated.

In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20-24 of gestation.

Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled and individual embryos resulted in the successful derivation of putative EGC lines from Days 20 to 24 with high efficiency.

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells.

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration.

Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.

Objectives: We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells.

Methods: The exocrine pellet fraction was cultivated in suspension followed by monolayer culture.

EGF-induced proliferation of adult human pancreatic duct cells is mediated by the MEK/ERK cascade.

Human postnatal pancreatic duct cells are a potential source of new beta cells. Factors regulating proliferation of human pancreatic duct cells in vitro are unknown. In several other cell types, this process is influenced by ligands of the ErbB receptor family.

Expression of Wnt, Frizzled, sFRP, and DKK genes in adult human pancreas.

Wnts are important signaling molecules involved in many normal developmental processes in the human body as well as some forms of cancer. Nineteen Wnt genes are found in the human genome, as well as 10 Wnt receptor genes called Frizzled. Two coreceptors called LRP 5 and 6 are critical for Wnt signal transduction.

Nestin is expressed in vascular endothelial cells in the adult human pancreas.

In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion.