LipoMag Transfection Kit

Fat distribution differences between males and females are a major risk factor for metabolic disease, but their genetic etiology remains largely unknown. Here, we establish ADGRG6 as a major factor in adipogenesis and gender fat distribution. Deletion of ADGRG6 in human adipocytes impairs adipogenesis due to reduced cAMP signaling. Conditionally knocking out Adgrg6 in mouse adipocytes or deleting an intronic enhancer associated with gender fat distribution generates males with female-like fat deposition, which are protected against high-fat-diet-induced obesity and have improved insulin response. To showcase its therapeutic potential, we demonstrate that CRISPRi targeting of the Adgrg6 promoter or enhancer prevents high-fat-diet-induced obesity. Combined, our results associate ADGRG6 as a gender fat distribution gene and highlight its potential as a therapeutic target for metabolic disease.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. Antioxidant enzymes decrease the generation of ionizing radiation (IR)-induced free radicals and therefore are associate to radioresistance. The main goal of this work is to study the involvement of peroxiredoxin-2 (Prx2) in the radio and chemoradiotherapy response in CRC cells in vitro and in vivo. We found that Prx2 oxidation state is associated to differential response to ionizing radiation in CRC cell lines. HCT116 radioresistant CRC cell line have lower ROS levels and a higher monomer/dimer Prx2 ratio, compared to halfway resistant Caco-2 and T84, and radiosensitive LoVo cell line. Constitutive and transient Prx2 silencing in CRC cells increase ROS levels, and most importantly, enhance in vitro radiation sensitivity. In addition, we showed that administration of IR plus oxaliplatin in down regulated Prx2 HCT116 cells has higher citotoxic effect than in control cells. Finally, radiosensitizing effect of Prx2 depletion was confirmed in vivo. These results suggest that Prx2 is an important component in tumoral radiation response, and their inhibition could improve radio and chemoradiotherapy protocols in patients with CRC.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that processes DNA-repair function and controls cellular response to oxidative stress. Endothelial progenitor cells (EPCs) are recruited to oxidative stress-rich injured vascular walls and positively contribute to vascular repair and endothelialization. We hypothesized that APE1 functions for EPCs-mediated inhibition of neointima formation in injured vasculature. EPCs isolated from bone marrow cells of C57BL6 mice (12-16 wk old) were able to survive in the presence of hydrogen peroxide (H2O2; up to 1,000 μM) due to the highly expressed reactive oxygen species (ROS) scavengers. However, adhesion capacity of EPCs was significantly inhibited by H2O2 (100 μM) even though an intracellular ROS was retained at small level. An APE1-selective inhibitor or RNA interference-mediated knockdown of endogenous APE1 in EPCs aggravated the H2O2-mediated inhibition of EPCs-adhesion. In contrast, when APE1 was overexpressed in EPCs using an adenovirus harboring the APE1 gene (APE-EPCs), adhesion was significantly improved during oxidative stress. To examine in vivo effects of APE1 in EPCs, APE-EPCs were transplanted via the tail vein after wire-mediated injury of the mouse femoral artery. The number of adherent EPCs at injured vascular walls and the vascular repair effect of EPCs were enhanced in APE-EPCs compared with control EPCs. Among the cellular functions of EPCs, adhesion is especially sensitive to oxidative stress. APE1 enhances in vivo vascular repair effects of EPCs in part through the maintenance of adhesion properties of EPCs. APE1 may be a novel and useful target gene for effective cellular transplantation therapy.

Genetically modified pigs play an important role in agriculture and biomedical research; hence, new efficient methods are needed to obtain genetically engineered cells and animals. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system represents an effective genome editing tool. It consists of two key molecules: single guide RNA (sgRNA) and the Cas9 endonuclease that can be introduced into the cells as one plasmid. Typical delivery methods for CRISPR/Cas9 components are limited by low transfection efficiency or toxic effects on cells. Here, we describe the use of magnetic nanoparticles and gradient magnetic field to improve delivery of CRISPR/Cas9 constructs into porcine fetal fibroblasts. Polyethylenimine-coated nanoparticles with magnetic iron oxide core were used to form magnetic plasmid DNA lipoplexes. CRISPR/Cas9 construct was prepared to induce site-specific cutting at the porcine H11 locus. Quantitative assessment of genomic cleavage by sequence trace decomposition demonstrated that the magnetofection efficiency was more than 3.5 times higher compared to the classic lipofection method. The Tracking of Indels by Decomposition web tool precisely determined the spectrum of indels that occurred. Simultaneously, no additional cytotoxicity associated with the utilization of magnetic nanoparticles was observed. Our results indicate that magnetofection enables effective delivery of the CRISPR/Cas9 construct into porcine fetal fibroblasts with low cell toxicity.

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by rapid growth and extensive invasiveness. Overexpression of insulin-like growth factor-binding protein-2 (IGFBP-2) has been reported in GBM. However, it remains to be determined how IGFBP-2 is involved in the progression of GBM. We utilized short hairpin-RNA (shRNA) expression retroviral vectors to inactivate the IGFBP-2 gene permanently in two human GBM cell lines, U251 and YKG-1. The stable knockdown of IGFBP-2 resulted in decreased invasiveness, decreased saturation density of the cells in vitro, and decreased tumorigenicity in nude mice. Transcriptional profiling of both lines revealed several genes that were significantly down-regulated by inactivation of IGFBP-2. One such gene was CD24, which has been implicated in progression of various cancers. Indeed, CD24 was expressed in most GBM cases and the inactivation of CD24 in GBM cells suppressed cellular invasiveness, as was the case for IGFBP-2. Forced overexpression of CD24 led to increased invasiveness of both IGFBP-2-inactivated GBM cell lines and also A172, a human GBM cell line with low endogenous CD24. Further supporting the inter-relationship between IGFBP-2 and CD24, knockdown of IGFBP-2 suppressed the CD24 promoter activity. Moreover, both CD24 promoter activity and in vitro invasiveness were restored in knockdown cells by transfection with an IGFBP-2 expression plasmid. These results indicate that CD24 is modulated by IGFBP-2 and contributes to IGFBP-2-enhanced invasiveness of GBM cells.