Calcium Phosphate Transfection Research Articles

Chondroitin sulfate modified calcium phosphate nanoparticles for efficient transfection via caveolin-mediated endocytosis

Efficient transfection remains a challenge for gene delivery in both cell biological scientific research and gene therapeutic fields. Existing transfection strategies rarely pay attention to altering the endocytosis pathway of nanocarriers for transfection efficiency improvement. In this work, we innovatively postulated that calcium phosphate nanoparticles coated with glycosaminoglycan could be internalized by cells mainly through caveolin-mediated endocytosis pathway allowing genes to bypass lysosome route, and hence enhance the transfection efficiency. To achieve this, we developed calcium phosphate nanoparticles (CP-ALN-CS) coated with chondroitin sulfate (CS) and alendronate (ALN) in a modular manner. The CP-ALN-CS had a hydrodynamic size of 131.0 ± 8.7 nm and exhibited favorable dispersity, stability, and resistance to nuclease degradation. Unlike conventional calcium phosphate and PEI-based transfection, CP-ALN-CS exhibited efficient cellular uptake with co-localization in Golgi apparatus and endoplasmic reticulum. Through bypassing the lysosome involved cellular uptake route, CP-ALN-CS can effectively protect genes from degradation and relieve cytotoxicity. After loading plasmid DNA, CP-ALN-CS showed extraordinary transfection efficiency in HEK 293T cells, outperforming the PEI which is considered as the gold standard. The current work provides a novel and facile approach to improve gene transfection efficiency and is valuable for the design of next-generation in vitro transfection reagents.

Anti-aging Effects of Alu Antisense RNA on Human Fibroblast Senescence Through the MEK-ERK Pathway Mediated by KIF15.

To investigate whether human short interspersed nuclear element antisense RNA (Alu antisense RNA; Alu asRNA) could delay human fibroblast senescence and explore the underlying mechanisms. We transfected Alu asRNA into senescent human fibroblasts and used cell counting kit-8 (CCK-8), reactive oxygen species (ROS), and senescence-associated beta-galactosidase (SA-β-gal) staining methods to analyze the anti-aging effects of Alu asRNA on the fibroblasts. We also used an RNA-sequencing (RNA-seq) method to investigate the Alu asRNA-specific mechanisms of anti-aging. We examined the effects of KIF15 on the anti-aging role induced by Alu asRNA. We also investigated the mechanisms underlying a KIF15-induced proliferation of senescent human fibroblasts. The CCK-8, ROS and SA-β-gal results showed that Alu asRNA could delay fibroblast aging. RNA-seq showed 183 differentially expressed genes (DEGs) in Alu asRNA transfected fibroblasts compared with fibroblasts transfected with the calcium phosphate transfection (CPT) reagent. The KEGG analysis showed that the cell cycle pathway was significantly enriched in the DEGs in fibroblasts transfected with Alu asRNA compared with fibroblasts transfected with the CPT reagent. Notably, Alu asRNA promoted the KIF15 expression and activated the MEK-ERK signaling pathway. Our results suggest that Alu asRNA could promote senescent fibroblast proliferation via activation of the KIF15-mediated MEK-ERK signaling pathway.

Сравнительный анализ эффективности общедоступных методов трансфекции модельных клеточных линий для задач биотехнологии

Transient gene expression is one of the most common methods in molecular biology, equally relevant for basic research projects and biotechnological industries. Despite the existence of commercial transfection systems, which afford high transfection efficiency and high expression levels of reporter genes, expanding such systems to industrial scales is often problematic due to high costs of the reagents. The well-described methods of cationic and calcium-phosphate transfection are accessible and ensure reproducible results at much lower costs. This study is aimed at comparative validation of calcium phosphate and cationic (polyethylenimine-based) transfection protocols along with the commercially available TurboFect reagent for mono- and cotransfections on a panel of commonly used cell lines including HEK293T, Huh7, BHK-21, CHO and MRC5. The efficiency of transfection with plasmid constructs encoding different fluorescent proteins was measured by flow cytometry. Of all the tested methods, calcium phosphate transfection afforded the highest efficiency of plasmid DNA delivery in all the cell lines except BHK21, for which the PEI method turned out to be more efficient than calcium phosphate transfection, and CHO, for which both methods showed comparable efficiency.

Preparation of Pseudo-typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity.

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Simultaneous tracking of capsid VP26, envelope protein gC localization in living cells infected with double fluorescent duck enteritis virus

Duck enteritis virus (DEV) can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of DEV vaccine strain pE1 (pDEV-EF1) has been constructed in our previous study. Based on pE1, a recombinant mutated clone pDL (pVP26CFP-gCRFP), which carries a red fluorescent protein (mRFP) gene fused to the viral envelope protein gC in combination with a cyan fluorescent protein (CFP) gene fused to the viral capsid VP26, was constructed by two-step Red/ET recombination and the recombinant virus rDL (rVP26CFP-gCRFP) was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate transfection. Western blot analysis revealed that VP26-CFP and gC-mRFP were both expressed in fusion forms in rDL-infected CEFs, and subcellular localization study showed that gC-mRFP was mainly localized in whole cell at 36, 48 h post infection (p.i.); and then mostly migrated to the cytoplasm after 60 h.p.i., ; whereas VP26-CFP was localized in the nucleus in all stages of virus infection. Additionally, viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells by transmission electron microscopy, indicating that exogenous gene insertion has no effect on virus assembly. This study has laid a foundation for visually studying localization, transportation of DEV capsid proteins and envelope glycoproteins as well as virus assembly, virion movement and virus-cell interaction.

Enhancing transient protein expression in HEK-293 cells by briefly exposing the culture to DMSO

BackgroundTransient expression of proteins in mammalian cells is a key technique for many functional and structural studies of human and higher eukaryotic genes as well as for the production of recombinant protein therapeutics. Maximizing the expression efficiency to achieve a higher expression yield is desirable and may be even critical when, for instance, an expressed protein must be characterized at the single-cell level. New MethodsOur goal was to develop a simple method by which protein expression yield in human embryonic kidney (HEK)-293 cells could be enhanced with a brief treatment of dimethyl sulfoxide (DMSO) solution. ResultsBy expressing green fluorescent protein (GFP) as a reporter protein using the calcium phosphate transfection method and imaging a large population of cells, we found that a 5-min exposure of 10 % DMSO to HEK-293 cells, 4 h after transfection of the protein of interest, leads to ∼1.6-fold increase in the expression yield without causing any appreciable cytotoxicity. By expressing an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and separately a kainate receptor in HEK-293 cells and measuring glutamate-induced whole-cell current response, we also found that such a brief DMSO treatment did not affect channel activity. ConclusionThis method is simple, efficient and inexpensive to use for enhancing transient transfection yield in HEK-293 cells.

Decompression Process of Glycerol Shock Treatment Can Overcome Endo-Lysosomal Barriers for Intracellular Delivery.

The glycerol shock treatment has been used to improve the calcium phosphate transfection efficacy for several decades because of its high effectiveness and low toxicity. However, the mechanism of glycerol shock treatment is still obscure. In this study, the endo-lysosomal leakage assay demonstrated that the decompression process of glycerol shock treatment could enhance endo-lysosomal membrane permeabilization, which resulted in facilitating endo-lysosomal escape for effective intracellular delivery. The enhanced decompression treatment derived from glycerol shock treatment could increase the change of osmotic pressure further, which showed higher efficacy for intracellular delivery. Herein, we speculated that the endo-lysosomal swelling originated from the decompression process of glycerol shock treatment could cause endo-lysosomal damage.

Measuring Changes in Keap1-Nrf2 Protein Complex Conformation in Individual Cells by FLIM-FRET.

The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-mediated stress response is a major cellular defense mechanism against endogenous and exogenous oxidants, electrophiles, and pro-inflammatory agents. A number of Nrf2 inducers are being developed to therapeutically stimulate this pathway. Inducers are typically sensed by Kelch-like ECH-associated protein 1 (Keap1), a negative regulator and a binding partner of Nrf2. Modifications of Keap1 by oxidants or electrophiles, or its targeting by compounds that disrupt its interaction with Nrf2, alter the conformation of the Keap1-Nrf2 protein complex, which initiates the accumulation of Nrf2 required for mounting a stress response. To detect conformational changes in the Keap1-Nrf2 complex in live cells, we have developed a procedure based on Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer (FLIM-FRET). The procedure includes a FLIM time course in cells expressing fluorescently-tagged Nrf2 and Keap1, followed by an extended analysis pipeline that quantifies changes in fluorescence lifetime of labeled Nrf2. The analysis visualizes and removes intensity-dependent bias in fluorescence lifetime measured with the Time-Correlated Single Photon Counting (TCSPC) approach, thereby improving the accuracy of quantification. The throughput is increased by the whole-experiment analysis within the newly developed FLIM dataset tool (FLIMDAST) and by the time-lapse FLIM described here. This pipeline is also suitable for applications beyond the Nrf2 field that assess small changes in fluorescence lifetime of objects with variable fluorescence intensities measured using TCSPC-based FLIM. © 2020 The Authors. Basic Protocol 1: Lipofectamine 2000 transfection Alternate Protocol 1: Calcium phosphate transfection Basic Protocol 2: Time course with individual FLIM Alternate Protocol 2: Time course with time-lapse FLIM Support Protocol: Measuring Instrument Response Function (IRF) Basic Protocol 3: Data analysis in SPCImage Basic Protocol 4: Data processing in ImageJ/FIJI Basic Protocol 5: Experiment analysis in FLIMDAST.

YAP promotes endothelial barrier repair by repressing STAT3/VEGF signaling

AimsEndothelial barrier dysfunction is associated with multiple diseases, and barrier repair may be a possible therapeutic target. Yes-associated protein and its pathway have been implicated in organ repair after injury. However, the mechanisms underlying barrier repair and any role YAP plays in the process are unclear. This study aimed to explore the role and mechanism of YAP in the repair of endothelial cell permeability after TNF-α-induced injury. Main methodsA trans-endothelial electrical resistance assay was performed to investigate changes in endothelial cell permeability. Lentivirus packaging by calcium phosphate transfection was used to construct endothelial cell lines with knocked down or overexpressed YAP. Western blotting, immunofluorescence, CO-IP, and real-time PCR were used to detect related protein and gene expression. Key findingsYAP is involved in the repair process of TNF-α-induced endothelial cell permeability injury; its overexpression promotes repair of endothelial cell permeability, and knockdown weakens repair ability. Moreover, YAP may promote repair by down-regulating STAT3 activity, thereby inhibiting VEGF expression. SignificanceElucidating the role of YAP in endothelial cell permeability repair process after injury might reveal mechanisms of endothelial barrier repair and provide therapeutic targets for treatment of vascular hyper-permeability disease.

Optimization and Characterization of Calcium Phosphate Transfection in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a promising tool for cell therapy, and gene-modified MSCs further expand their applications. To take full advantage of MSCs as a therapeutic approach, developing effective gene transfer methods is critical. Calcium phosphate transfection is well-established and safe, but the protocols need to be optimized according to different cell types. Currently, there is no optimized protocol for MSCs. This study optimized the protocol of calcium phosphate transfection for MSCs and highlighted the importance of serum during the process of transfection. More interestingly, the behavior of gene overexpression in MSCs in the in vivo environment was verified.

Construction and identification of recombinant lentiviral plasmid of receptor for advanced glycation end products

Objective To construct a recombinant lentiviral plasmid of receptor for advanced glycation end products (RAGE) and establish RAGE stable expressing hepatocellular carcinoma cell models in order to explore the pathogenesis of RAGE in the occurrence and development of hepatocellular carcinoma. Methods RAGE gene fragments were amplified by PCR and cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro. Then the recombinant lentiviral plasmid and packaging plasmid were used to package lentivirus in HEK293T cells by calcium phosphate transfection. Lentivirus supernatant was collected to infect hepatoma cells HepG2 and Huh7. After that cells were screened by puromycin. Finally, RAGE expression was detected by real-time PCR and Western blot. Results RAGE gene fragment was successfully amplified and inserted into lentiviral expression vector. After packaging the lentivirus-infected liver cancer cells, real-time quantitative PCR and Western blot results showed that the mRNA (P<0.05) and protein expression of RAGE cells stably expressing RAGE were significantly higher than that of the control cells. Conclusions The RAGE overexpressing hepatocarcinoma cell line was successfully constructed by using lentiviral expression vector, which laid a good foundation for further study on the pathogenesis of RAGE. Key words: Liver neoplasms; Lentiviral expression vector; Receptor for advanced glycation end products

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40V/mm, 20μs each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION)+6-DMAP+CHX (12.07%) activation was higher than that of ION+6-DMAP (8.6%) activation or ION+CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION+6-DMAP+CHX and DC+6-DMAP+CHX groups. The blastocyst rate of ION+6-DMAP+CHX-activated oocytes in the basic rabbit culture medium (M-199)+10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199+10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.

Влияние диметилсульфоксидного шока на эффективность кальций-фосфатной трансфекции клеток

Влияние диметилсульфоксидного шока на эффективность кальций-фосфатной трансфекции клеток

Efficacy of recombinant adenovirus expressing a fusion gene from GM-CSF and Epstein-Barr virus LMP2A in a mouse tumor model

ABSTRACT In this study, purified GM-CSF and LMP2A mRNAs were amplified by PCR. Then, the GM-CSF and LMP2A sequences were connected by the polypeptide linker (Gly4Ser)3 using gene splicing by overlap extension. The constructed fusion gene GC2A was inserted into the adenovirus vector. Then the recombinant vector was introduced into HEK 293T cells by calcium phosphate transfection to package the adenovirus. The levels of antibodies against the GM-CSF and LMP2Afusion proteins were measured by ELISA, and the CTL activity of the mouse splenic lymphocytes was determined by lactate dehydrogenase (LDH) release assay. Immunotherapy of mouse tumor (EBV-positive epithelial tumor cell line (GT39)) tissues was performed, and their morphologies were assessed. Finally, the data of each group were analyzed using SPSS 11.5 statistical software. The recombinant adenovirus could replicate in HEK 293Tcells and induce humoral and cellular immune responses in the mice. The maximum dose resulted in an antibody titer of 18500 (184.5 ± 8.7 pg/ml). At an effector: target ratio of 40:1, maximum specific lysis was observed which was approximately three times that detected in the control immunized mice. The tumor inhibition rate was approximately 76% compared with the control groups, indicating the presence of significant differences among the groups. Tumor-infiltrating lymphocytes were detected by hematoxylin-eosin (HE) staining. The recombinant adenovirus induced humoral and cellular immune responses and inhibited tumor growth in mice. It provided a theoretical basis and candidate vaccine for further preclinical trials.

Transfecting CHO-K1 Cells: Comparison of CaPO4, Electroporation and Lipoplex Method with In-house Prepared Polyplex

The extensive demand of large scale therapeutic protein production in Chinese Hamster Ovarian cell lines increased the need of developing efficient and cost effective transfection reagent. Polyethylenimine has recently emerged as an attractive alternative to currently employed conventional transfection methods. In the present report, we attempted to develop inexpensive and easy to prepare polyethylenimine-based transfection reagent and compared its transfection efficiency with calcium phosphate, electroporation and lipoplex mediated transfection methods. Our results indicated highest transfection efficiency with in-house prepared polyplexes as marked by maximum percentage of enhanced cyan fluorescence protein positive cells. These findings contribute towards the development of economical and effective solution for high protein expression in Chinese hamster ovarian system.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons.

In order to characterize genetically encoded tools under the most relevant conditions, the constructs need to be expressed in the cell type in which they will be used. This is a major hurdle in developing optogenetic tools for neuronal cells, due to the difficulty of gene transfer to these cells. Several protocols have been developed for transfecting neurons, focusing on improved transfection efficiency. However, obtaining healthy cells is as important. We monitored transfected cell health by measuring electrophysiological parameters, and used them as a guideline to optimize transfection. Here we describe an optimized transfection protocol that achieves reasonably high efficiency (10-20 %) with no discernable impact on cell health, as characterized by electrophysiology.

P50: Detection of COP1-interacting proteins using co-immunoprecipitation followed MALDI-TOF mass spectrometry

In 2004, COP1 was characterized as p53 E3-specific human ubiquitin ligase according to in vitro studies. Later, oncogenic activity of COP1 and correlation of its overexpression with level of p53 protein in clinical tumor samples and cell lines were shown. High expression level of COP1 was described in 25 of 32 cases of breast carcinomas and in 76 out of 171 cases of ovarian carcinomas. High expression level of COP1 was described in hepatocellular carcinoma cell lines (PLC, Hep3B and HepG2, Huh7). In 40 of 55 cases of pancreatic cancer, overexpression of COP1 was also noted. Some types of leukemia, melanoma, breast, lung and prostate cancer contain focal deletions of COP1. COP1 overexpression was also found in non- oncological diseases like Duchenne muscular dystrophy, ischemic cardiomyopathy and juvenile dermatomyositis. Similar to other p53-related E3 -ubiquitin ligases COP1 has different substrates. The selection of target for ubiquitination depends on cell type or differentiation stage. Among the substrates of COP1, besides p53 are: c-Jun, ETV1, ACC, TORC2, FOXO1 and C/EBPα, FIP200. To identify novel proteins that interact with COP1 we applied proteomics. Proteins co-immunoprecipitated with COP1 were analyzed by MALDI -TOF mass spectrometry. To achieve this, COP1–3xFlag expression vector was generated and transfected into HEK 293T cells by calcium phosphate transfection method. Protein complexes were immunoprecipitated with anti-FLAG beads. Bound proteins were then separated by 1D SDS–PAGE gel electrophoresis and analyzed by means of ESI-LC/MS/MS mass spectrometry. Subsequent bioinformatics analysis of the interacting proteins was employed. About 25% of identified proteins were attributed to the cytoskeleton proteins, almost 20% of proteins had known role in metabolism. According to the literature data, it was not surprising to find several proteins involved in the lipid metabolism. Finally, a significant groups of interactants play role in transcription, cell adhesion, apoptosis, protein modification and transport. It is important to note that several COP1-bound proteins can be strong regulators of cell cycle in G2/M transition. Our data can be used for the subsequent studies of previously unknown roles of COP1 in different diseases. This work was funded by Grants from Russian Scientific Foundation – Russia (No. 114-15-00816 ) and Grant of Molecular and Cellular Biology of the Presidium of the Russian Academy of Sciences.