Shortening the biologics clinical timeline with a novel method for generating stable, high-producing cell pools and clones.

Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the coronavirus disease 2019 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substances produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the Chinese hamster ovary cell line genome. This allows for rapid production of high-expressing stable cell pools and clones that reach titers of 4-12 g/l in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarities in glycosylation profiles.

MicroRNA profiling in the mouse hypothalamus reveals oxytocin-regulating microRNA.

Oxytocin (Oxt), produced in the hypothalamic paraventricular and supraoptic nuclei for transport to and release from the posterior pituitary, was originally discovered through its role in lactation and parturition. Oxt also plays important roles in the central nervous system by influencing various behaviors. MicroRNAs (miRNAs), endogenous regulators of many genes, are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. We performed miRNA expression profiling of the mouse hypothalamus by deep sequencing. Among the sequenced and cross-mapped small RNAs, expression of known miRNAs and unknown miRNAs candidates were analyzed. We investigated in detail one miRNA, miR-24, and found that it is a novel regulator of Oxt and controls both transcript and peptide levels of Oxt. These results provide insights into potential neurohypophysial hormone regulation mediated by miRNAs.

Investigating the in vivo expression patterns of miR-7 microRNA family members in the adult mouse brain.

MicroRNAs (miRNAs) are endogenous non-coding RNAs that play critical roles in regulating transcription in eukaryotes. miRNAs are involved in a wide-range of biological processes. Because miRNA research is relatively new, it is not surprising that tools and techniques to study their biology are still being developed. Our laboratory is interested in carefully delineating the in vivo expression patterns between three closely related miR-7 miRNA family members in the mouse. The miR-7 family of miRNAs is conserved between humans and invertebrates. The miR-7 family also shows conserved expression in neural tissues, and members of this family are involved in the development of sensory structures in flies, associated with many types of cancer, and likely play a role in osmoregulation in the mouse brain. In this study, we used multiple methods to differentiate the expression of the miR-7 family members in the mouse brain. Our results suggest that although there are useful tools for exploring miRNA expression in vivo, better tools and/or methods are still needed for thorough in vivo studies of closely related family members.

Wnt signaling stimulates transcriptional outcome of the Hedgehog pathway by stabilizing GLI1 mRNA.

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/beta-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity. The newly described mode of regulation of GLI1 seems to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells.

Zebrafish zic2a patterns the forebrain through modulation of Hedgehog-activated gene expression.

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. We demonstrate that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. We show that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance our understanding of a key gene regulatory network that, when disrupted, causes HPE.

A novel role for zebrafish zic2a during forebrain development.

Patterns of transcription factor expression establish a blueprint for the vertebrate forebrain early in embryogenesis. In the future diencephalon, several genes with patterned expression have been identified, yet their specific functions and interactions between them are not well understood. We have uncovered a crucial role for one such gene, zic2a, during formation of the anterior diencephalon in zebrafish. We show that zic2a is required for transcription of the prethalamic markers arx and dlx2a. This function is required during early steps of prethalamic development, soon after its specification. zic genes are evolutionarily related to glis, transcription factors that mediate hedgehog signaling. Intriguingly, the hedgehog signaling pathway also acts to promote development of the prethalamus. We asked if zic2a interacts with hedgehog signaling in the context of forebrain development in zebrafish. Our data show that hedgehog signaling and zic2a function at different times, and therefore act in parallel pathways during forebrain development. Taken together, our results identify Zic2a as a novel regulator of prethalamic development, and show that it functions independently of hedgehog signaling.