STEM-25. ADENO-ASSOCIATED VIRUS INTRAVENTRICULAR INJECTION ALLOWS THE RESTRICTED TRANSDUCTION OF GLIOBLASTOMA CELLS NESTED IN THE PERI- AND SUB-VENTRICULAR ZONE IN A PATIENT-DERIVED ORTHOTOPIC XENOGRAFT MODEL

INTRODUCTION: Glioblastoma (GBM) seems to arise from cells nested in the subventricular zone (SVZ), a neurogenic zone in the adult brain. After striatal engraftment of patient-derived GBM cells in mice, we showed that some tumor cells escape the tumor mass to colonize the SVZ. We aimed to determine if the SVZ-nested GBM cells could be highlighted by the intraventricular injection of a viral vector. MATERIALS AND METHODS: We determined the optimal Adeno-Associated Virus (AAV) serotype to transduce patient-derived GBM cells cultured in 3D (GB1). We studied the spreading of the virus 4 weeks after intraventricular injection in mice. We realized striatal engraftment with GB1 cells, previously transfected with a lentiviral construction in order to express the Red-Fluorescent-Protein (RFP) spontaneously, while they express Green-Fluorescent-Protein (eGFP) only in presence of Cre-recombinase. 10 weeks after engraftment, an AAV expressing Cre-Recombinase was injected in the lateral ventricle and mice were perfused at 14 weeks. RESULTS: AAV serotype DJ (AAVDJ) efficiently transduced GB1 cells. 4 weeks after intraventricular injection, the AAVDJ transduced cells in the SVZ and the medial part of the caudoputamen (mCP). The median of the longest distance between the right ventricle and the transduced cells in the mCP was 293.8µm [245 – 376.5]. In 5 mice, the median of the shortest distance between RFP-positive GBM cells and right ventricle was 580.1µm [535 – 785.1]. SVZ was not colonized and eGFP signal was not found. In the 4 other mice, SVZ was invaded and eGFP signal was detected. The median of the longest distance between the right ventricle and eGFP-positive tumor cells was 240.5µm [195.7 – 372.5]. The median of the eGFP volume was 9 499 404µm3. CONCLUSION: It is possible to transduce GBM cells nested in the peri- and sub-ventricular zone thanks to AAV intraventricular injection.

NUDT16L1 (TIRR) knockdown increases genome editing efficacy

Введение. Эффективная точная коррекция мутаций очень важна для внедрения CRISPR-Cas в качестве инструмента для потенциальной генной терапии. Механизм направленной гомологичной репарации (НГР), активируемый после внесения нуклеазой двунитевого разрыва, позволяет корректировать все существующие типы мутаций. Однако НГР не является доминирующим механизмом в клетке, что ограничивает ее эффективность. В нашем исследовании впервые описано, как фактор репарации NUDT16L1 может контролировать эффективность НГР при редактировании с помощью CRISPR-Cas. Цель: исследовать влияние нокдауна и гиперэкспрессии NUDT16L1 на эффективность НГР. Методы. Исследование проводили на клеточной культуре HEK293T. Систему CRISPR-Cas в плазмиде, а также вектор для гиперэкспрессии NUDT16L1 доставляли путём липофекции. Для нокдауна гена NUDT16L1 использовали малые интерферирующие РНК. Результаты. Нокдаун NUDT16L1 повышает уровень НГР в плазмидном и геномном локусах, что проявляется повышением доли GFP-позитивных клеток в 1,8-3,6 раз. Заключение. Нокдаун гена NUDT16L1 может быть использован для повышения эффективности исправления патогенных вариантов ДНК методом геномного редактирования. Introduction. Effective precise knock-in is crucial for implementing CRISPR-Cas9 system as an efficient instrument for potential gene therapy. Homology directed repair (HDR) pathway allows correction of all types of existing mutations. However, HDR is not a major repair pathway of the cell that limits its efficiency. In our study, we present for the first time how repair factors NUDT16L1 controls HDR efficiency. Aim: to study an influence of NUDT16L1 knockdown and overexpression on the HDR efficacy. Methods. HEK293T culture was used to perform the research. Plasmid CRISPR-Cas system along with NUDT16L1 overexpression vector were delivered with lipofection. For NUDT16L1 knockdown small interfering RNAs were used. Results. We discovered that knockdown of NUDT16L1 enhances HDR both in the plasmid and genomic loci increasing eGFP signal from 1.8 to 3.6 times in HEK293T cells. Conclusion. NUDT16L1 knockdown could be used for enhancing of the pathogenic mutations correction through genome editing.

EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells

The erythropoietin receptor (EpoR) has been traditionally thought as an erythroid specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In the present study we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice in which very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further demonstrated by the EpoR-tdTomato-Cre mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles of EPO/EpoR in hematopoiesis which warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

198 Germline-Specific Expression of the Murine Oct4-EGFP Transgene in the Pig

The Oct4 gene is crucial for undisturbed early embryonic development and maintenance of pluripotency in the mouse. It is found in mouse pre-implantation embryos after embryonic genome activation. After gastrulation, expression is restricted to germ cells. Limited research has been performed on OCT4 expression in the domestic pig, which is a valuable large animal model in biomedicine. Previously, we generated Oct4-EGFP reporter pigs carrying the genomic sequence of the murine Oct4 gene fused to the EGFP cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev. 20, 1563-1575, 10.1089/scd.2010.0399). In the present study, we used this animal model to analyse the expression profile of the murine Oct4-EGFP transgene in porcine oocytes, in vivo-derived embryos (4-cell embryos, 8- to 16-cell embryos, morulae, and blastocysts) and ovaries. We studied whether the murine Oct4-EGFP transgene mimics the expression pattern of the endogenous OCT4 protein in transgenic pigs. Immature oocytes were isolated from ovaries of Oct4-EGFP transgenic sows (n = 5) using slicing methods. For collection of porcine embryos, wild-type sows were inseminated with sperm from an Oct4-EGFP transgenic boar. Sows were sacrificed 3, 4, and 5 days after insemination, and embryos were recovered by flushing oviducts and uterus and analysed by confocal microscopy. Ovaries obtained from female animals (5–12 months) were enzymatically dissociated and analysed using flow cytometry. Immature oocytes (n = 19) showed a very low, diffuse EGFP signal in cytoplasm. Embryos up to the 4-cell stage (n = 45) did not show Oct4-EGFP transgene expression. For the first time, EGFP fluorescence was detected at the 8-cell stage (n = 29) and a strong EGFP signal was observed in 16-cell stages and morulae (n = 53). In blastocysts from Day 5 (n = 40) EGFP fluorescence was not restricted to the inner cell mass (ICM), but was also seen in the trophectoderm (TE). Expression of EGFP was not detected in ovarian cells (n = 12). Thereafter, we analysed the expression pattern of endogenous OCT4 protein by immunostaining in nontransgenic porcine oocytes and pre-implantation embryos. As in Oct4-EGFP transgenic embryos, no expression of OCT4 was observed in 4-cell embryos (n = 12). Nuclear staining first became visible at the 8-cell stage (n = 12), with a strong signal observed in 16-cell stages and morulae (n = 18). In blastocysts from Day 5 (n = 26), both ICM and TE cell nuclei showed expression of OCT4 protein. These results demonstrate that the Oct4-EGFP transgene expression pattern reproduces the endogenous OCT4 protein expression profile in porcine oocytes and pre-implantation embryos. The Oct4-EGFP transgene was first detected at the 8-cell stage, consistent with embryonic genome activation, which is initiated at the 4-cell stage. However, Oct4-EGFP expression was not detected in ovarian cells. This might be related to the very low expression pattern of the Oct4-EGFP transgene in primary oocytes. In summary, the Oct4-EGFP transgene in the pig provides a useful marker for monitoring pluripotency in pre-implantation embryos after embryonic genome activation. In ongoing experiments, we are analysing the expression profile of the Oct4-EGFP transgene and endogenous OCT4 protein in porcine pre-implantation embryos from Days 8 and 11.

Improved Golgi-like Visualization in Retrogradely Projecting Neurons after EGFP-Adenovirus Infection in Adult Rat and Monkey

An adenovirus vector was generated using a neuron-specific promoter synapsin I and enhanced green fluorescent protein (EGFP) reporter (AdSynEGFP). In addition, two modifications were identified that resulted in robust and reliable retrograde transport and EGFP expression after injection of the virus into three different brain regions in adult rats (medial prefrontal cortex, posterior thalamic nuclear group, and CA1). These are post-injection survival times of 14 days and addition of high concentrations of NaCl (≥600 mM) to the injection buffer. These modifications resulted in obvious improvement in the intensity of the EGFP signal and in the number of labeled cells. Use of anti-EGFP in immunofluorescence or immunoperoxidase processing further enhanced the signal so that Golgi-like filling of dendritic spines and axon collaterals was routinely achieved. Effectiveness of the AdSynEGFP for Golgi-like filling was confirmed in one rhesus monkey with injections in visual area V4. Because of the long-term viability of the infected neurons (at least up to 28 days in rats and 22 days in monkey), this AdSynEGFP is suitable for use in microcircuitry studies in combination with other fluorescently tagged elements, including anterogradely labeled extrinsic projections. The native EGFP signal (without antibody enhancement) may be sufficient for studies involving cultured cells or slices. (J Histochem Cytochem 54:539-548, 2006)