An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).

Time course of transient expression of pre-α-pro-GDNF and pre-ß-pro-GDNF transcripts, and mGDNF mRNA region in Krushinsky-Molodkina rat brain after audiogenic seizures.

The expression of glial cell line-derived neurothrophic factor (GDNF) transcript forms pre-(α)pro-gdnf, pre-(ß)pro-gdnf, and their common region m-gdnf in the pons as well as the inferior (IC) and superior colliculi in Krushinsky-Molodkina (KM) rats and in the strain "0" was analyzed by quantitative real-time polymerase chain reaction (PCR) in the control (unstimulated KM and "0" rats) and 1.5, 4.5, and 8 h after auditory stimulation. Such stimulation induced audiogenic seizures (AS) in KM rats. Audiogenic seizure was not observed in "0" rats, which was obtained by selection for the absence of AS in a population of F2 hybrids between KM and Wistar rats not predisposed to AS. A significant drop in the level of all transcripts was observed 1.5 h after auditory stimulation in both KM and "0" rats. In most cases, the average expression of α and ß isoforms and m-region 4.5 h after stimulation was greater than those after 1.5 and 8 h. At the same time, the expression of pre-(ß)pro-gdnf in the IC of KM rats 4.5 h after the stimulation was significantly lower than after 1.5 or 8 h. This work presents the first demonstration of different time courses of expression of the α and ß GDNF isoforms during physiological processes in genotype-specific pathology.

Reparative properties of human glioblastoma cells after single exposure to a wide range of X-ray doses.

Radiation therapy induces double-stranded DNA breaks in tumor cells, which leads to their death. A fraction of glioblastoma cells repair such breaks and reinitiate tumor growth. It was necessary to identify the relationship between high radiation doses and the proliferative activity of glioblastoma cells, and to evaluate the contribution of DNA repair pathways, homologous recombination (HR), and nonhomologous end joining (NHEJ) to tumor-cell recovery. We demonstrated that the GO1 culture derived from glioblastoma cells from Patient G, who had previously been irradiated, proved to be less sensitive to radiation than the Sus\fP2 glioblastoma culture was from Patient S, who had not been exposed to radiation before. GO1 cell proliferation decreased with radiation dose, and MTT decreased to 35% after a single exposure to 125 Gγ. The proliferative potential of glioblastoma culture Sus\fP2 decreased to 35% after exposure to 5 Gγ. At low radiation doses, cell proliferation and the expression of RAD51 were decreased; at high doses, cell proliferation was correlated with Ku70 protein expression. Therefore, HR and NHEJ are involved in DNA break repair after exposure to different radiation doses. Low doses induce HR, while higher doses induce the faster but less accurate NHEJ pathway of double-stranded DNA break repair.

Availability of Pre- and Pro-regions of Transgenic GDNF Affects the Ability to Induce Axonal Sprout Growth.

Plasmids containing four GFP-tagged isoforms of the human GDNF gene, with both pre- and pro-regions (pre-pro- GDNF), with the pre- (pre-GDNF) or the pro-region (pro-GDNF) alone, and without the pre- and pro-regions (mGDNF), were used to transfect HEK293 cells (human embryonic kidney cell line). The effect of the transgenic products on the growth of processes was studied in the spinal ganglia of 14-day rat embryos. Media conditioned by the transgenic cells were used to culture explants and dissociated cells of embryonic dorsal root ganglia attached to the bottom of the plate. Medium conditioned by gfp-transgenic HEK293 cells was used as the control. Spinal ganglia explants and dissociated cells cultured in a medium supplemented with recombinant GDNF (recGDNF) as well as in conditioned media containing the pre-GDNF and mGDNF products demonstrated active growth of processes immunopositive for neuronal marker beta-3-tubulin as early as on culture day 4. The ganglia and cells cultured in control medium and media conditioned by cells transgenic for pro-GDNF had no or very few processes even after 10 days of culture.