Design of transfections: Implementation of design of experiments for cell transfection fine tuning.

Transfection is the process by which nucleic acids are introduced into eukaryotic cells. This is fundamental in basic research for studying gene function and modulation of gene expression as well as for many bioprocesses in the manufacturing of clinical-grade recombinant biologics from cells. Transfection efficiency is a critical parameter to increase biologics' productivity; the right protocol has to be identified to ensure high transfection efficiency and therefore high product yield. Design of experiments (DoE) is a mathematical method that has become a key tool in bioprocess development. Based on the DoE method, we developed an operational flow that we called "Design of Transfections" (DoT) for specific transfection modeling and identification of the optimal transfection conditions. As a proof of principle, we applied the DoT workflow to optimize a cell transfection chemical protocol for neural progenitors, using polyethyleneimine (PEI). We simultaneously varied key influencing factors, namely concentration and type of PEI, DNA concentration, and cell density. The transfection efficiency was measured by fluorescence imaging followed by automatic counting of the green fluorescent transfected cells. Taking advantage of the DoT workflow, we developed a new simple, efficient, and economically advantageous PEI transfection protocol through which we were able to obtain a transfection efficiency of 34%.

The Transcription Regulator Patz1 Is Essential for Neural Stem Cell Maintenance and Proliferation.

Proper regulation of neurogenesis, the process by which new neurons are generated from neural stem and progenitor cells (NS/PCs), is essential for embryonic brain development and adult brain function. The transcription regulator Patz1 is ubiquitously expressed in early mouse embryos and has a key role in embryonic stem cell maintenance. At later stages, the detection of Patz1 expression mainly in the developing brain suggests a specific involvement of Patz1 in neurogenesis. To address this point, we first got insights in Patz1 expression profile in different brain territories at both embryonic and postnatal stages, evidencing a general decreasing trend with respect to time. Then, we performed in vivo and ex vivo analysis of Patz1-knockout mice, focusing on the ventricular and subventricular zone, where we confirmed Patz1 enrichment through the analysis of public RNA-seq datasets. Both embryos and adults showed a significant reduction in the number of Patz1-null NS/PCs, as well as of their self-renewal capability, compared to controls. Consistently, molecular analysis revealed the downregulation of stemness markers in NS/PCs derived from Patz1-null mice. Overall, these data demonstrate the requirement of Patz1 for NS/PC maintenance and proliferation, suggesting new roles for this key transcription factor specifically in brain development and plasticity, with possible implications for neurodegenerative disorders and glial brain tumors.

Role of PA2G4P4 pseudogene in bladder cancer tumorigenesis.

BACKGROUND: Many pseudogenes possess biological activities and play important roles in the pathogenesis of various types of cancer including bladder cancer (BlCa), which still lacks suitable molecular biomarkers. Recently, pseudogenes were found to be significantly enriched in a pan-cancer classification based on the Cancer Genome Atlas gene expression data. Among them, the top-ranking pseudogene was the proliferation-associated 2G4 pseudogene 4 (PA2G4P4). METHODS: Genomic and transcript features of PA2G4P4 were determined by GeneBank database analysis followed by 5' RACE experiments. Therefore, we conducted a retrospective molecular study on a cohort of 45 patients of BlCa. PA2G4P4 expression was measured by RT-qPCR, whereas PA2G4P4 transcript distribution was analyzed by in situ hybridization on both normal and cancerous histological sections and compared to the immunolocalization of its parental PA2G4/EBP1 protein. Finally, we tested the effects of PA2G4P4 depletion on proliferation, migration, and death of BlCa cells. RESULTS: We showed for the first time PA2G4P4 overexpression in BlCa tissues and in cell lines. PA2G4P4 distribution strictly overlaps PA2G4/EBP1 protein localization. Moreover, we showed that PA2G4P4 knockdown affects both proliferation and migration of BlCa cells, highlighting its potential oncogenic role. CONCLUSIONS: PA2G4P4 may play a functional role as an oncogene in BlCa development, suggesting it as a good candidate for future investigation and new clinical applications.

TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation.

Pax8 and TTF-1 are transcription factors involved in the morphogenesis of the thyroid gland and in the transcriptional regulation of thyroid-specific genes. Both proteins are expressed in few tissues but their simultaneous presence occurs only in the thyroid where they interact physically and functionally allowing the regulation of genes that are markers of the thyroid differentiated phenotype. TAZ is a transcriptional coactivator that regulates the activity of several transcription factors therefore playing a central role in tissue-specific transcription. The recently demonstrated physical and functional interaction between TAZ and TTF-1 in the lung raised the question of whether TAZ could be an important regulatory molecule also in the thyroid. In this study, we demonstrate the presence of TAZ in thyroid cells and the existence of an important cooperation between TAZ and the transcription factors Pax8 and TTF-1 in the modulation of thyroid gene expression. In addition, we reveal that the three proteins are co-expressed in the nucleus of differentiated thyroid cells and that TAZ interacts with both Pax8 and TTF-1, in vitro and in vivo. More importantly, we show that this interaction leads to a significant enhancement of the transcriptional activity of Pax8 and TTF-1 on the thyroglobulin promoter thus suggesting a role of TAZ in the control of genes involved in thyroid development and differentiation.