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mljgoums 2024, 18(5): 34-38 |
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Daneshparvar A, Jamhiri I, Razban V, Fallahi J, Hamidizadeh N, Moghtaderi B et al . Engineering a DYRK1B R102C mutation: insights into metabolic syndrome pathogenesis through lentiviral gene delivery. mljgoums 2024; 18 (5) :34-38
URL: http://mlj.goums.ac.ir/article-1-1763-en.html
URL: http://mlj.goums.ac.ir/article-1-1763-en.html
Afrooz Daneshparvar1 
, Iman Jamhiri2 , Vahid Razban3 , Jafar Fallahi3 , Nasrin Hamidizadeh2 , Behnam Moghtaderi4 , Mehdi Dianatpour5
, Iman Jamhiri2 , Vahid Razban3 , Jafar Fallahi3 , Nasrin Hamidizadeh2 , Behnam Moghtaderi4 , Mehdi Dianatpour5
1- Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ; Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
2- Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
4- Department of Pathobiology, Shiraz Veterinary Medicine, Shiraz University, Shiraz, Iran
5- Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Human Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran ,mdianatpur@gmail.com
2- Molecular Dermatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
4- Department of Pathobiology, Shiraz Veterinary Medicine, Shiraz University, Shiraz, Iran
5- Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; Department of Human Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran ,
Keywords: DYRK1B protein, human, Metabolic syndrome, Mutagenesis, Cloning, Organism, Lentivirus, Genetic vectors
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Results
Mutagenesis by overlap extention PCR
The results showed that the desired mutation was performed correctly using the OE-PCR method. Following the primary PCR reactions, the secondary PCR reactions were produced in multiple bands, including the expected band, which was gel-excised and sent for sequencing. These reaction results are shown in Figure 3. DNA sequencing indicated that the point mutation was correctly performed.
Discussion
Today, gene transfer is one of the essential techniques in diagnosing, preventing and treating diseases under gene therapy. Different vectors are used for gene transfer. Among the viral vectors, lentiviruses have gained attention in recent years, considering their outstanding characteristics and important implications for clinical trials. As essential tools to investigate the molecular pathways involved in the disease, they are being developed and can be helpful in the design of the disease model (39-41). These viruses belong to the retroviridae family and, like retroviruses, can insert their genome into the host's genome after entering the host cell (37,41-43). Can do this during the cell division phase. Nevertheless, lentiviruses can enter the host cell's nucleus and insert their genome into the host's genome without requiring cell division (37,42,44,45). Many unnecessary and pathogenic genes of the virus have been removed, and DNA replication defects increase biological safety in these vectors (24,43).
Second-generation lentiviral vectors are composed of three separate lentiviral vectors. One of which in this study is the leGO-iG2 transfer vector, and the rest are lentiviral packaging vectors (pMD2 and psPAX2). In the present study, after being mutated in the mouse DYRK1B, it was cloned in the leGO-iG2 transfer vector containing the GFP marker and then transfected with the pMD2 and psPAX2 vectors in HEK 293T cells to produce lentivirus in these cells. After observing the green light resulting from the GFP expression in HEK 293T cells under a fluorescent microscope, recombinant lentivirus was confirmed, and virus titration was performed by flow cytometry as well.
This study is based on the production of recombinant lentivirus, which is achieved by inserting a gene into its genome, and we described the technique of producing recombinant lentivirus carrying the mutated gene. The use of recombinant lentivirus carrying DYRK1B R102C to produce disease models in animals, including mice, can be very useful in discovering the molecular mechanisms and pathways involved in metabolic syndrome.
DYRK1B overexpression has been found to promote cell cycle progression in certain cancers (46-49), and it has been suggested that DYRK1B protein plays a vital role in pathways that are disrupted in MetS (19,50-53). The molecular mechanisms of this event are not very clear. However, it seems that Dyrk1b protein inhibits the rate of glucose uptake and glycolysis by inhibiting the Ras-RAF-MEK pathway. Further, it has been found that the R102C mutation augments p27Kip turnover and enhances the adipogenic effects of DYRK1B (54). We acknowledge that the limitation of this study is that several other options for stages of lentivirus production were not investigated in this study. However, the primary purpose of this study was to identify steps that can be easily replicated in most laboratories when working with hard-to-transfect cells without having to purchase any additional equipment or incur extra costs. It would have been better to investigate other mutations involved in the function of the DYRK1B, such as the H90P missense mutation. For this, more cost and time were required, which we hope will be done by us or others in future research. Considering the advantages of lentiviruses in vivo use, one of our limitations is the lack of in vivo use of these lentiviruses. In the future, our focus is on using these lentiviruses to create transgenic eukaryotic cells and MetS disease models. It is necessary to mention that, according to the goals of lentivirus production containing the DYRK1B R102C mutation, which is transferred to eukaryotic cells, and finally, production of the disease model, we did not evaluate immune responses to lentivirus or lentivirus components in this study, but it should be noted that cell turnover in vitro is not representative of an in vivo setting.
This lentivirus was used in the present study to express the DYRK1B-R102C stably. There are no reports on lentiviral production for genes involved in MetS. Lentiviral vector-based cell therapy is safe in human and animal experiments. Therefore, this method is essential for treating patients with MetS.
Conclusion
We have described a method that can be successfully used for lentiviral gene transfer into eukaryotic cells. Lentiviruses carrying the DYRK1B R102C mutation offer significant advantages for in vitro and in vivo research on metabolic syndrome. The use of lentiviruses can be a strong approach to developing gene therapy and treating rare and incurable diseases.
Acknowledgement
The authors thank the Human Genetics Department of Shiraz University of Medical Sciences laboratory staff. This research was funded by Shiraz University of Medical Sciences (Grant No. 11216) as part of a Ph.D. thesis.
Funding sources
The authors of this article did not receive any financial support for the research.
Ethical statement
Not applicable.
Conflicts of interest
The authors declare no conflicts of interest.
Author contributions
All authors have contributed equally.
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Introduction
Metabolic syndrome (MetS) is determined by a set of multiple physical diseases that concertedly increase diabetes mellitus, cardiovascular disease, and vascular and neurological complications such as cerebrovascular accidents (1-4). According to the World Health Organization (WHO), blood pressure, obesity, and lipid disorders are essential components of MetS (5-7). This syndrome is accompanied by a decrease in the life quality of patients and causes serious damage to the healthcare system (8,9). It should be noted that MetS has become a global health problem. The prevalence of MetS worldwide reaches 20% to 30% according to increases with age and by gender (10-12).
The DYRK1B gene is involved in some diseases, such as MetS. Missense mutations in the DYRK1B are associated with MetS. An arginine to cysteine substitution at position 102 (R102C) in the highly conserved domain alters the protein's function (13-15). MetS is a rare autosomal dominant disorder caused by two missense mutations, H90P and R102C, in DYRK1B located on chromosome q13.219. MetS is characterized by abdominal obesity, type 2 diabetes, hypertension, and premature coronary artery disease (16-18). According to the results, the mutant alleles increase the DYRK1B function. DYRK1B-H90P or DYRK1B-R102C overexpression in HepG2 hepatocytes results in greater induction of the gluconeogenesis enzyme (Glucose-6-phosphatase) than normal DYRK1B. Additionally, R102C mutation affects the effect of DYRK1B on Hedgehog and Wnt signaling pathways and promotes the fat differentiation in 3T3-L1 preadipocyte cells (19-22).
Site-directed mutagenesis, one of the essential techniques in molecular biology, has been widely used to investigate the structure and function of nucleic acids and proteins, the mechanisms of genetic diseases, and the effect of genome modification (23-25). PCR-mediated site-directed mutagenesis is one of the most powerful methods for creating gene mutations in vitro (26-29). This method consists of two sequential steps of PCR. The primary PCR reactions produce two mutated DNA fragments with overlapping ends and a secondary reaction that creates a single piece by joining two components (30,31).
Lentiviral vectors are handy for investigating mutant gene expression. Lentiviruses are of interest considering their ability to infect dividing and non-dividing cells, enter the cell genome, explore gene and protein function, low level of cellular toxicity, and their application in gene therapy and cell therapy (32-35). Therefore, their unique characteristics make them suitable for investigating the expression of the desired mutant gene in the in vitro/in vivo environment (36-38).
Currently, gene therapy using lentivirus is being performed in a wide range of diseases. The purpose of this study is to describe the scientific steps of engineering recombinant lentivirus carrying DYRK1B-R102C and placing it in the transgenic pathway. The production of such a lentivirus can help in creating MetS models and more comprehensive investigations of this disease.
Methods
Study design
The Shiraz University of Medical Sciences, which follows the National Institutes of Health guidelines for the care and use of animals, approved the protocols and experiments in this study. In the present fundamental study, RNA samples were extracted from the fat tissues of 3-month-old mice free of the pathogen to create mutations in the desired gene. Considering the higher expression of DYRK1B in adipose tissues, samples were prepared from the adipose tissues around the mice’s testicles. Then, RNA was extracted from the samples and used for cDNA synthesis. To isolate adipose tissues, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100/10 mg/kg). A small incision was made in the abdomen with a sterile scissor, and the fat mass around the testicles was removed with the help of sterile forceps. The steps for separating adipose tissue from mice are shown in Figure 1.
RNA isolation and cDNA synthesis
TriPure Isolation Reagent (Roche, Germany) accomplished the RNA extraction from mouse fat tissue. Optical density ratios were examined using 260/280 nm; the extraction quality was assessed using a Nanodrop™ spectrophotometer (Nanodrop; Thermo Fisher Scientific, Wilmington, DE, USA). The cDNA was synthesized using 3.0 μg total RNA by RevertAid™ F first S and cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). In brief, the reaction was performed with 3.0 μg total RNA, 4 μl 5x reaction buffer, 1 μl oligo-dT or random hexamer primers, 2μl dNTP mix, 1 μl reverse transcriptase, and 1ml Ribolock inhibitor. The reaction conditions were as follows: 25 °C for 5 minutes, 42 °C for 60 minutes, 70 °C for 5 minutes, and the cDNA was stored at -80 °C.
Primer design
The gene-specific primers (GSP), including forward (FGSP) and reverse (RGSP), were designed to contain restriction sites NotIand EcoRI by AllelID v.7.5 software (PREMIER Biosoft, Palo Alto, CA, USA). The forward mutagenic primers (FMP) and reverse mutagenic primers (RMP) were complementary to each other, with the mutagenic bases (CT) in the center of each primer. The primer sequence is shown in Table 1.
Metabolic syndrome (MetS) is determined by a set of multiple physical diseases that concertedly increase diabetes mellitus, cardiovascular disease, and vascular and neurological complications such as cerebrovascular accidents (1-4). According to the World Health Organization (WHO), blood pressure, obesity, and lipid disorders are essential components of MetS (5-7). This syndrome is accompanied by a decrease in the life quality of patients and causes serious damage to the healthcare system (8,9). It should be noted that MetS has become a global health problem. The prevalence of MetS worldwide reaches 20% to 30% according to increases with age and by gender (10-12).
The DYRK1B gene is involved in some diseases, such as MetS. Missense mutations in the DYRK1B are associated with MetS. An arginine to cysteine substitution at position 102 (R102C) in the highly conserved domain alters the protein's function (13-15). MetS is a rare autosomal dominant disorder caused by two missense mutations, H90P and R102C, in DYRK1B located on chromosome q13.219. MetS is characterized by abdominal obesity, type 2 diabetes, hypertension, and premature coronary artery disease (16-18). According to the results, the mutant alleles increase the DYRK1B function. DYRK1B-H90P or DYRK1B-R102C overexpression in HepG2 hepatocytes results in greater induction of the gluconeogenesis enzyme (Glucose-6-phosphatase) than normal DYRK1B. Additionally, R102C mutation affects the effect of DYRK1B on Hedgehog and Wnt signaling pathways and promotes the fat differentiation in 3T3-L1 preadipocyte cells (19-22).
Site-directed mutagenesis, one of the essential techniques in molecular biology, has been widely used to investigate the structure and function of nucleic acids and proteins, the mechanisms of genetic diseases, and the effect of genome modification (23-25). PCR-mediated site-directed mutagenesis is one of the most powerful methods for creating gene mutations in vitro (26-29). This method consists of two sequential steps of PCR. The primary PCR reactions produce two mutated DNA fragments with overlapping ends and a secondary reaction that creates a single piece by joining two components (30,31).
Lentiviral vectors are handy for investigating mutant gene expression. Lentiviruses are of interest considering their ability to infect dividing and non-dividing cells, enter the cell genome, explore gene and protein function, low level of cellular toxicity, and their application in gene therapy and cell therapy (32-35). Therefore, their unique characteristics make them suitable for investigating the expression of the desired mutant gene in the in vitro/in vivo environment (36-38).
Currently, gene therapy using lentivirus is being performed in a wide range of diseases. The purpose of this study is to describe the scientific steps of engineering recombinant lentivirus carrying DYRK1B-R102C and placing it in the transgenic pathway. The production of such a lentivirus can help in creating MetS models and more comprehensive investigations of this disease.
Methods
Study design
The Shiraz University of Medical Sciences, which follows the National Institutes of Health guidelines for the care and use of animals, approved the protocols and experiments in this study. In the present fundamental study, RNA samples were extracted from the fat tissues of 3-month-old mice free of the pathogen to create mutations in the desired gene. Considering the higher expression of DYRK1B in adipose tissues, samples were prepared from the adipose tissues around the mice’s testicles. Then, RNA was extracted from the samples and used for cDNA synthesis. To isolate adipose tissues, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100/10 mg/kg). A small incision was made in the abdomen with a sterile scissor, and the fat mass around the testicles was removed with the help of sterile forceps. The steps for separating adipose tissue from mice are shown in Figure 1.
RNA isolation and cDNA synthesis
TriPure Isolation Reagent (Roche, Germany) accomplished the RNA extraction from mouse fat tissue. Optical density ratios were examined using 260/280 nm; the extraction quality was assessed using a Nanodrop™ spectrophotometer (Nanodrop; Thermo Fisher Scientific, Wilmington, DE, USA). The cDNA was synthesized using 3.0 μg total RNA by RevertAid™ F first S and cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). In brief, the reaction was performed with 3.0 μg total RNA, 4 μl 5x reaction buffer, 1 μl oligo-dT or random hexamer primers, 2μl dNTP mix, 1 μl reverse transcriptase, and 1ml Ribolock inhibitor. The reaction conditions were as follows: 25 °C for 5 minutes, 42 °C for 60 minutes, 70 °C for 5 minutes, and the cDNA was stored at -80 °C.
Primer design
The gene-specific primers (GSP), including forward (FGSP) and reverse (RGSP), were designed to contain restriction sites NotIand EcoRI by AllelID v.7.5 software (PREMIER Biosoft, Palo Alto, CA, USA). The forward mutagenic primers (FMP) and reverse mutagenic primers (RMP) were complementary to each other, with the mutagenic bases (CT) in the center of each primer. The primer sequence is shown in Table 1.
.PNG)
.PNG)
Mutagenesis by overlap extension-PCR
Mutagenesis by OE-PCR consists of two primary PCR reactions and a secondary PCR reaction. The direct PCR reaction for each primer pair was carried out in tubes of separate, using 10 μl 2x Master Mix Red (Ampliqon A/S, Odense, Denmark), 1 μl FGSP, 1 μl RMP, 1 μl cDNA, 7 μl water, and another tube consist of 10 μl Mastermix, 1 μl RGSP,1 μl FMP,1 μl cDNA, 7 μl water. The PCR profile was used as follows: initial denaturation for 5 minutes at 95 °C, followed by 30 cycles of 30 seconds at 94 °C, 30 seconds at 60 °C, and 1 minute at 72 °C. A final elongation of 72 °C followed this profile for 5 minutes. PCR reactions were performed using the Applied Biosystems Veriti 96-Well Thermal Cycler Instrument (ABI, Veriti, USA). At 60 °C for all primers, set the annealing temperature (Ta). Subsequently, 1% agarose gel stained with a safe stain visualized the primary PCR products. Gel excise and purify the PCR products, then for the overlap PCR, using a 1:1 ratio of both excised fragments (~75ng each) in the master mix without the primers for the first 10-13 cycles. Later the forward primer of the first fragment and the reverse primer of the last piece were added to the cocktail and ran for another 25-30 cycles, which produced the full-length mutagenic DNA and then excised gel-purified to be used for cloning.
Cloning and transformation
The mutagenic DNA and LeGO-iG2 plasmid were digested with EcoRI and NotI (Fermentas, Germany). These enzymes have restriction sites in LeGO-iG2 and the prepared mutagenic DNA. The digestion reaction was performed using 5 μl of DYRK1B DNA (212 ng/μl), 1μl of tango buffer, 1 μl of EcoRI (10 U/μl), 1 μl of NotI (10 U/μl), and 2 μl of DDW. In another reaction LeGO-iG2 2.5 µl (487 ng/μl), tango buffer 1 μl, EcoRI 1μl (10 U/μl), NotI 1 μl (10 U/μl), and DDW 2 μl.
The digested products were purified from the gel using the gel extraction kit (Qiagen, USA). The ligation reaction was carried out as follows: 20μl of DYRK1B, 7μl of LeGO-iG2, 3.3μl of T4 DNA ligase buffer, 1.7 μl of T4 DNA ligase (5 U/μl) and incubate at 16 °C for 20 hours. 10 μl of ligation reaction in 70 µL of competent Escherichia coli DH5 α were transformed using the heat shock method. The transformed bacteria were cultured on LB agar containing ampicillin 100 μg/mL and incubated overnight at 37 °C. Colony-PCR was carried out to approve colonies containing the recombinant vector. In the next step, the recombinant vector was extracted by a QIAprep Miniprep Kit (Qiagen, USA). The cloning accuracy was confirmed by enzyme digestion with EcoRI (10 U/μl) and NotI (10 U/μl), followed by sequencing (DNAMAN software). Enzymatic digestion was carried out using recombinant vector 5 μl (238 ng/μl), 1 μl of Buffer, 1 μl of NotI (10 U/μl), 1μl of EcoRI (10 U/μl), and DDW 3 μl.
Virus production and titration
A lentivirus-based vector expressing GFP was produced using transient co-transfection of Hek-293T cells with a three-plasmid combination. In brief, (i) transfer lentiviral vector 15µg (LeGO-iG2), (ii) envelope plasmid VSV-G 10µg (psPAX2), and (iii) packaging plasmid 10µg (pMD2) were used in a calcium phosphate transfection mix and transfected into human embryonic kidney cells (HEK 293T cells) (Pasteur Institute, Iran) and culture medium was replaced to fresh medium 18 hours after the transfection and viral supernatants were harvested 36-, 48- and 72-hours post-transfection. We centrifuged at low speed (300 g for 5 minutes) and filtered through a 0.45 m filter. The lentiviruses supernatant was concentrated by ultra-centrifugation at 70,000 g in a swinging bucket rotor for 1 hour at 4 °C. The viral pellet was then re-suspended in 200 μl of PBS 1X. Titration of the eGFP-expressing lentivirus vector was carried out by HEK 293T cell transduction, and the eGFP-expressing cell was examined by flow cytometry (BD Biosciences). The lentivirus stocks were diluted in four concentrations (10X, 100X, 1,000X, and 10,000X), and then these dilutions were added to a four-well plate with 2 × 105 293T cells per well. GFP-positive cells were estimated by flow cytometry. The titration results by flow cytometry are shown in Figure 2. Using the formula: TU/ml = seeding cell number × fluorescent cell number percentage × dilution factor/transduction volume in ml, Transducing Units (TU) were calculated.
Mutagenesis by OE-PCR consists of two primary PCR reactions and a secondary PCR reaction. The direct PCR reaction for each primer pair was carried out in tubes of separate, using 10 μl 2x Master Mix Red (Ampliqon A/S, Odense, Denmark), 1 μl FGSP, 1 μl RMP, 1 μl cDNA, 7 μl water, and another tube consist of 10 μl Mastermix, 1 μl RGSP,1 μl FMP,1 μl cDNA, 7 μl water. The PCR profile was used as follows: initial denaturation for 5 minutes at 95 °C, followed by 30 cycles of 30 seconds at 94 °C, 30 seconds at 60 °C, and 1 minute at 72 °C. A final elongation of 72 °C followed this profile for 5 minutes. PCR reactions were performed using the Applied Biosystems Veriti 96-Well Thermal Cycler Instrument (ABI, Veriti, USA). At 60 °C for all primers, set the annealing temperature (Ta). Subsequently, 1% agarose gel stained with a safe stain visualized the primary PCR products. Gel excise and purify the PCR products, then for the overlap PCR, using a 1:1 ratio of both excised fragments (~75ng each) in the master mix without the primers for the first 10-13 cycles. Later the forward primer of the first fragment and the reverse primer of the last piece were added to the cocktail and ran for another 25-30 cycles, which produced the full-length mutagenic DNA and then excised gel-purified to be used for cloning.
Cloning and transformation
The mutagenic DNA and LeGO-iG2 plasmid were digested with EcoRI and NotI (Fermentas, Germany). These enzymes have restriction sites in LeGO-iG2 and the prepared mutagenic DNA. The digestion reaction was performed using 5 μl of DYRK1B DNA (212 ng/μl), 1μl of tango buffer, 1 μl of EcoRI (10 U/μl), 1 μl of NotI (10 U/μl), and 2 μl of DDW. In another reaction LeGO-iG2 2.5 µl (487 ng/μl), tango buffer 1 μl, EcoRI 1μl (10 U/μl), NotI 1 μl (10 U/μl), and DDW 2 μl.
The digested products were purified from the gel using the gel extraction kit (Qiagen, USA). The ligation reaction was carried out as follows: 20μl of DYRK1B, 7μl of LeGO-iG2, 3.3μl of T4 DNA ligase buffer, 1.7 μl of T4 DNA ligase (5 U/μl) and incubate at 16 °C for 20 hours. 10 μl of ligation reaction in 70 µL of competent Escherichia coli DH5 α were transformed using the heat shock method. The transformed bacteria were cultured on LB agar containing ampicillin 100 μg/mL and incubated overnight at 37 °C. Colony-PCR was carried out to approve colonies containing the recombinant vector. In the next step, the recombinant vector was extracted by a QIAprep Miniprep Kit (Qiagen, USA). The cloning accuracy was confirmed by enzyme digestion with EcoRI (10 U/μl) and NotI (10 U/μl), followed by sequencing (DNAMAN software). Enzymatic digestion was carried out using recombinant vector 5 μl (238 ng/μl), 1 μl of Buffer, 1 μl of NotI (10 U/μl), 1μl of EcoRI (10 U/μl), and DDW 3 μl.
Virus production and titration
A lentivirus-based vector expressing GFP was produced using transient co-transfection of Hek-293T cells with a three-plasmid combination. In brief, (i) transfer lentiviral vector 15µg (LeGO-iG2), (ii) envelope plasmid VSV-G 10µg (psPAX2), and (iii) packaging plasmid 10µg (pMD2) were used in a calcium phosphate transfection mix and transfected into human embryonic kidney cells (HEK 293T cells) (Pasteur Institute, Iran) and culture medium was replaced to fresh medium 18 hours after the transfection and viral supernatants were harvested 36-, 48- and 72-hours post-transfection. We centrifuged at low speed (300 g for 5 minutes) and filtered through a 0.45 m filter. The lentiviruses supernatant was concentrated by ultra-centrifugation at 70,000 g in a swinging bucket rotor for 1 hour at 4 °C. The viral pellet was then re-suspended in 200 μl of PBS 1X. Titration of the eGFP-expressing lentivirus vector was carried out by HEK 293T cell transduction, and the eGFP-expressing cell was examined by flow cytometry (BD Biosciences). The lentivirus stocks were diluted in four concentrations (10X, 100X, 1,000X, and 10,000X), and then these dilutions were added to a four-well plate with 2 × 105 293T cells per well. GFP-positive cells were estimated by flow cytometry. The titration results by flow cytometry are shown in Figure 2. Using the formula: TU/ml = seeding cell number × fluorescent cell number percentage × dilution factor/transduction volume in ml, Transducing Units (TU) were calculated.
.PNG) Figure 2. The lentirivus titration by flow cytometry. A) GFP analysis on HEK293T cells after the transduction with different concentrations of lentirivus (10X, 100X, 1,000X, and 10,000X). B) The GFP positive percentage. |
Results
Mutagenesis by overlap extention PCR
The results showed that the desired mutation was performed correctly using the OE-PCR method. Following the primary PCR reactions, the secondary PCR reactions were produced in multiple bands, including the expected band, which was gel-excised and sent for sequencing. These reaction results are shown in Figure 3. DNA sequencing indicated that the point mutation was correctly performed.
.PNG) Figure 3. The mutagenesis overlaps extension PCR product. A) Both primary PCR reactions produced single bands estimated at 540bp and 1,700bp, which matched the expected size. Since single bands were obtained, gel purification was unnecessary. B) The secondary PCR reaction produced multiple bands, including the expected band, at 2,240 bp (Indicated by black arrow). The gel purification had to be done because the single band was not created. DNA ladder, 1 Kb. |
Recombinant vector production
The mutagenic DNA was electrophoresed after digestion with EcoRI and NotI. A fragment without any considerable size change was observed. The LeGO iG2 vector was also digested with the same enzymes, and a 7829-bp band was observed. In the next step, the target gene was inserted into the LeGO-iG2 using T4 DNA ligase. The ligation product was used to transform Escherichia coli DH5, and colony-PCR was carried out using the forward and reverse primers for DYRK1B to confirm gene insertion into LeGO-iG2. The colony-PCR product was electrophoresed in gel, and a fragment of 2240 bp was observed. The recombinant vector was purified using the QIAprep Miniprep Kit and subjected to digestion with EcoRI and NotI (i), the 7829 bp band was identical to the linearized vector, and (ii) observed two pieces were. Another band was consistent with DYRK1B (2240 bp band). The DYRK1B 2240 bp band is shown in Figure 4. The extracted vectors were sent for sequencing, and the results were checked with BLAST. Successful cloning was finally confirmed by sequencing the recombinant vector, and the sequencing output is shown in Figure 5.
The mutagenic DNA was electrophoresed after digestion with EcoRI and NotI. A fragment without any considerable size change was observed. The LeGO iG2 vector was also digested with the same enzymes, and a 7829-bp band was observed. In the next step, the target gene was inserted into the LeGO-iG2 using T4 DNA ligase. The ligation product was used to transform Escherichia coli DH5, and colony-PCR was carried out using the forward and reverse primers for DYRK1B to confirm gene insertion into LeGO-iG2. The colony-PCR product was electrophoresed in gel, and a fragment of 2240 bp was observed. The recombinant vector was purified using the QIAprep Miniprep Kit and subjected to digestion with EcoRI and NotI (i), the 7829 bp band was identical to the linearized vector, and (ii) observed two pieces were. Another band was consistent with DYRK1B (2240 bp band). The DYRK1B 2240 bp band is shown in Figure 4. The extracted vectors were sent for sequencing, and the results were checked with BLAST. Successful cloning was finally confirmed by sequencing the recombinant vector, and the sequencing output is shown in Figure 5.
.PNG) Figure 4. The colony-PCR was performed to confirm colonies. After DNA extraction from the bacterial colonies, the PCR reaction was performed to confirm the presence of the DYRK1B R102C gene, sample 1 colony-PCR (2240 bp band). Sample 2, 1 Kb DNA ladder. .PNG) Figure 5. Sequencing to confirm cloning. A) Sequencing chromatogram with forward primer to confirm the accuracy of cloned gene fragment in LeGO-iG2 vector. B) Sequencing chromatogram showing the mutations as dots and highlighted sections in the aligned sequence. |
Virus production and titration
HEK 293T cell line was co-transfected with the plasmids leGO-iG2-DYRK1B, pMD2, and psPAX2 to produce a GFP-DYRK1B expressing lentiviral. Fluorescent microscopy showed that GFP was expressed 48 hours post-transfection in more than 90% of these cells Figure 6. Lentiviral-containing supernatant was concentrated using the ultracentrifuge-based method. The flow cytometry for viral titer measuring was performed 72- hours post-transduction of HEK 293T cells. Calculate virus titration using GFP positive cell percentage in 10,000X concentration (5%). The viral titer was 108 TU/ml on HEK293T cells.
HEK 293T cell line was co-transfected with the plasmids leGO-iG2-DYRK1B, pMD2, and psPAX2 to produce a GFP-DYRK1B expressing lentiviral. Fluorescent microscopy showed that GFP was expressed 48 hours post-transfection in more than 90% of these cells Figure 6. Lentiviral-containing supernatant was concentrated using the ultracentrifuge-based method. The flow cytometry for viral titer measuring was performed 72- hours post-transduction of HEK 293T cells. Calculate virus titration using GFP positive cell percentage in 10,000X concentration (5%). The viral titer was 108 TU/ml on HEK293T cells.
.PNG) Figure 6. Lentivirus production and GFP-DYRK1B gene expression under fluorescent microscopy. A) HEK293T cells before transfection, B) HEK293T cells 48 hours post-transfection |
Discussion
Today, gene transfer is one of the essential techniques in diagnosing, preventing and treating diseases under gene therapy. Different vectors are used for gene transfer. Among the viral vectors, lentiviruses have gained attention in recent years, considering their outstanding characteristics and important implications for clinical trials. As essential tools to investigate the molecular pathways involved in the disease, they are being developed and can be helpful in the design of the disease model (39-41). These viruses belong to the retroviridae family and, like retroviruses, can insert their genome into the host's genome after entering the host cell (37,41-43). Can do this during the cell division phase. Nevertheless, lentiviruses can enter the host cell's nucleus and insert their genome into the host's genome without requiring cell division (37,42,44,45). Many unnecessary and pathogenic genes of the virus have been removed, and DNA replication defects increase biological safety in these vectors (24,43).
Second-generation lentiviral vectors are composed of three separate lentiviral vectors. One of which in this study is the leGO-iG2 transfer vector, and the rest are lentiviral packaging vectors (pMD2 and psPAX2). In the present study, after being mutated in the mouse DYRK1B, it was cloned in the leGO-iG2 transfer vector containing the GFP marker and then transfected with the pMD2 and psPAX2 vectors in HEK 293T cells to produce lentivirus in these cells. After observing the green light resulting from the GFP expression in HEK 293T cells under a fluorescent microscope, recombinant lentivirus was confirmed, and virus titration was performed by flow cytometry as well.
This study is based on the production of recombinant lentivirus, which is achieved by inserting a gene into its genome, and we described the technique of producing recombinant lentivirus carrying the mutated gene. The use of recombinant lentivirus carrying DYRK1B R102C to produce disease models in animals, including mice, can be very useful in discovering the molecular mechanisms and pathways involved in metabolic syndrome.
DYRK1B overexpression has been found to promote cell cycle progression in certain cancers (46-49), and it has been suggested that DYRK1B protein plays a vital role in pathways that are disrupted in MetS (19,50-53). The molecular mechanisms of this event are not very clear. However, it seems that Dyrk1b protein inhibits the rate of glucose uptake and glycolysis by inhibiting the Ras-RAF-MEK pathway. Further, it has been found that the R102C mutation augments p27Kip turnover and enhances the adipogenic effects of DYRK1B (54). We acknowledge that the limitation of this study is that several other options for stages of lentivirus production were not investigated in this study. However, the primary purpose of this study was to identify steps that can be easily replicated in most laboratories when working with hard-to-transfect cells without having to purchase any additional equipment or incur extra costs. It would have been better to investigate other mutations involved in the function of the DYRK1B, such as the H90P missense mutation. For this, more cost and time were required, which we hope will be done by us or others in future research. Considering the advantages of lentiviruses in vivo use, one of our limitations is the lack of in vivo use of these lentiviruses. In the future, our focus is on using these lentiviruses to create transgenic eukaryotic cells and MetS disease models. It is necessary to mention that, according to the goals of lentivirus production containing the DYRK1B R102C mutation, which is transferred to eukaryotic cells, and finally, production of the disease model, we did not evaluate immune responses to lentivirus or lentivirus components in this study, but it should be noted that cell turnover in vitro is not representative of an in vivo setting.
This lentivirus was used in the present study to express the DYRK1B-R102C stably. There are no reports on lentiviral production for genes involved in MetS. Lentiviral vector-based cell therapy is safe in human and animal experiments. Therefore, this method is essential for treating patients with MetS.
Conclusion
We have described a method that can be successfully used for lentiviral gene transfer into eukaryotic cells. Lentiviruses carrying the DYRK1B R102C mutation offer significant advantages for in vitro and in vivo research on metabolic syndrome. The use of lentiviruses can be a strong approach to developing gene therapy and treating rare and incurable diseases.
Acknowledgement
The authors thank the Human Genetics Department of Shiraz University of Medical Sciences laboratory staff. This research was funded by Shiraz University of Medical Sciences (Grant No. 11216) as part of a Ph.D. thesis.
Funding sources
The authors of this article did not receive any financial support for the research.
Ethical statement
Not applicable.
Conflicts of interest
The authors declare no conflicts of interest.
Author contributions
All authors have contributed equally.
Research Article: Research Article |
Subject:
Molecular Medicine
Received: 2023/12/24 | Accepted: 2024/07/30 | Published: 2024/10/27 | ePublished: 2024/10/27
Received: 2023/12/24 | Accepted: 2024/07/30 | Published: 2024/10/27 | ePublished: 2024/10/27
References
1. Goh KK, Chen CY-A, Wu T-H, Chen C-H, Lu M-L. Crosstalk between schizophrenia and metabolic syndrome: The role of oxytocinergic dysfunction. International journal of molecular sciences. 2022; 23(13): 7092. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Bazmandegan G, Abbasifard M, Nadimi AE, Alinejad H, Kamiab Z. Cardiovascular risk factors in diabetic patients with and without metabolic syndrome: a study based on the Rafsanjan cohort study. Scientific Reports. 2023; 13(1): 559. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Farooqui AA, Farooqui T, Panza F, Frisardi V. Metabolic syndrome as a risk factor for neurological disorders. Cellular and Molecular Life Sciences. 2012; 69: 741-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Swarup S, Goyal A, Grigorova Y, Zeltser R. Metabolic syndrome. StatPearls [internet]: StatPearls Publishing; 2022. [View at Publisher] [PMID] [Google Scholar]
5. Alberti KGMM, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of a WHO consultation. Diabetic medicine. 1998; 15(7): 539-53.
https://doi.org/10.1002/(SICI)1096-9136(199807)15:7<539::AID-DIA668>3.0.CO;2-S [View at Publisher] [DOI] [Google Scholar]
6. Alberti KGM, Zimmet P, Shaw J. The metabolic syndrome-a new worldwide definition. The Lancet. 2005;366(9491):1059-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Goossens GH. The metabolic phenotype in obesity: fat mass, body fat distribution, and adipose tissue function. Obesity facts. 2017; 10(3): 207-15. [View at Publisher] [DOI] [PMID] [Google Scholar]
8. Naghipour M, Joukar F, Nikbakht H-A, Hassanipour S, Asgharnezhad M, Arab-Zozani M, et al. High prevalence of metabolic syndrome and its related demographic factors in North of Iran: results from the Persian Guilan cohort study. International Journal of Endocrinology. 2021;2021. [View at Publisher] [DOI] [PMID] [Google Scholar]
9. De Silva ST, Niriella MA, Ediriweera DS, Kottahachchi D, Kasturiratne A, de Silva AP, et al. Incidence and risk factors for metabolic syndrome among urban, adult Sri Lankans: a prospective, 7-year community cohort, follow-up study. Diabetology & Metabolic Syndrome. 2019;11(1):1-7. [View at Publisher] [DOI] [PMID] [Google Scholar]
10. Gunawan S, Aulia A, Soetikno V. Development of rat metabolic syndrome models: A review. Veterinary World. 2021;14(7):1774. [View at Publisher] [DOI] [PMID] [Google Scholar]
11. Pucci G, Alcidi R, Tap L, Battista F, Mattace-Raso F, Schillaci G. Sex-and gender-related prevalence, cardiovascular risk and therapeutic approach in metabolic syndrome: A review of the literature. Pharmacological research. 2017;120:34-42. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Dubey P, Singh V, Venishetty N, Trivedi M, Reddy SY, Lakshmanaswamy R, et al. Associations of sex hormone ratios with metabolic syndrome and inflammation in US adult men and women. Front Endocrinol (Lausanne). 2024;15:1384603. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. Bhat N, Narayanan A, Fathzadeh M, Shah K, Dianatpour M, Abou Ziki MD, et al. Dyrk1b promotes autophagy during skeletal muscle differentiation by upregulating 4e-bp1. Cellular Signalling. 2022; 90: 110186. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. Charkoftaki G, Thompson DC, Golla JP, Garcia-Milian R, Lam TT, Engel J, et al. Integrated multi-omics approach reveals a role of ALDH1A1 in lipid metabolism in human colon cancer cells. Chemico-biological interactions. 2019; 304: 88-96. [View at Publisher] [DOI] [PMID] [Google Scholar]
15. Keramati AR, Fathzadeh M, Singh R, Choi M, Faramarzi S, Mane S, et al. Identification of a novel disease gene for early onset atherosclerosis, diabetes and metabolic syndrome by whole exome sequencing and linkage analysis. Am Heart Assoc. 2013; 128(22). [View at Publisher] [DOI] [Google Scholar]
16. Abu Jhaisha S, Widowati EW, Kii I, Sonamoto R, Knapp S, Papadopoulos C, et al. DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain. 2017;7(1):6420. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. Mendoza-Caamal EC, Barajas-Olmos F, Mirzaeicheshmeh E, Ilizaliturri-Flores I, Aguilar-Salinas CA, Gómez-Velasco DV, et al. Two novel variants in DYRK1B causative of AOMS3: expanding the clinical spectrum. Orphanet journal of rare diseases. 2021; 16(1): 291. [View at Publisher] [DOI] [PMID] [Google Scholar]
18. Zhuang L, Jia K, Chen C, Li Z, Zhao J, Hu J, et al. DYRK1B-STAT3 drives cardiac hypertrophy and heart failure by impairing mitochondrial bioenergetics. Circulation. 2022;145(11):829-46. [View at Publisher] [DOI] [PMID] [Google Scholar]
19. Keramati AR, Fathzadeh M, Go GW, Singh R, Choi M, Faramarzi S, et al. A form of the metabolic syndrome associated with mutations in DYRK1B. The New England journal of medicine. 2014;370(20):1909-19. [View at Publisher] [DOI] [PMID] [Google Scholar]
20. Armanmehr A, Jafari Khamirani H, Zoghi S. Analysis of DYRK1B, PPARG, and CEBPB Expression Patterns in Adipose-Derived Stem Cells from Patients Carrying DYRK1B R102C and Healthy Individuals During Adipogenesis. 2022. [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Pinhas-Hamiel O. Type 2 Diabetes, Metabolic Syndrome and Lipid Metabolism. Yearbook of Pediatric Endocrinology 2014: Endorsed by the European Society for Paediatric Endocrinology (ESPE). 2014; 383: 171. [View at Publisher] [DOI] [Google Scholar]
22. Martin KA, Mani MV, Mani A. New targets to treat obesity and the metabolic syndrome. European journal of pharmacology. 2015; 763: 64-74. [View at Publisher] [DOI] [PMID] [Google Scholar]
23. Yang W, Jiang LH. Site-directed mutagenesis to study the structure-function relationships of ion channels. Methods in molecular biology (Clifton, NJ). 2013; 998: 257-66. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Zhang K, Yin X, Shi K, Zhang S, Wang J, Zhao S, et al. A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Scientific Reports. 2021;11(1):10454. [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Usman S, Bushaala A, Teh MT, Waseem A. Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction. Methods Mol Biol. 2024. [View at Publisher] [DOI] [PMID] [Google Scholar]
26. Liu H, Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC biotechnology. 2008; 8(1): 1-10. [View at Publisher] [DOI] [PMID] [Google Scholar]
27. Aiyar A, Xiang Y, Leis J. Site-directed mutagenesis using overlap extension PCR. In vitro mutagenesis protocols. 1996; 177-91. [View at Publisher] [DOI] [PMID] [Google Scholar]
28. Forloni M, Liu AY, Wajapeyee N. Creating insertions or deletions using overlap extension polymerase chain reaction (PCR) mutagenesis. Cold Spring Harbor Protocols. 2018;2018(8):pdb. prot097758. [View at Publisher] [DOI] [PMID] [Google Scholar]
29. Nelson MD, Fitch DH. Overlap extension PCR: an efficient method for transgene construction. Molecular methods for evolutionary genetics. 2011:459-70. [View at Publisher] [DOI] [PMID] [Google Scholar]
30. Alfranca A, Campanero MR, Redondo JM. New Methods for Disease Modeling Using Lentiviral Vectors. Trends in molecular medicine. 2018; 24(10): 825-37. [View at Publisher] [DOI] [PMID] [Google Scholar]
31. Tseng W-C, Lin J-W, Wei T-Y, Fang T-Y. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal Biochem. 2008; 375(2): 376-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
32. Ku M-W, Charneau P, Majlessi L. Use of lentiviral vectors in vaccination. Expert Review of Vaccines. 2021;20(12):1571-86. [View at Publisher] [DOI] [PMID] [Google Scholar]
33. Sakuma T, Barry MA, Ikeda Y. Lentiviral vectors: basic to translational. Biochemical Journal. 2012; 443(3): 603-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
34. McGinley L, McMahon J, Strappe P, Barry F, Murphy M, O'Toole D, et al. Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia. Stem cell research & therapy. 2011;2:1-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
35. Magnani A, Semeraro M, Adam F, Booth C, Dupré L, Morris E, et al. Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome. Nature Medicine. 2022; 28(1): 71-80. [View at Publisher] [DOI] [PMID] [Google Scholar]
36. Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia. 2018; 32(7): 1529-41. [View at Publisher] [DOI] [PMID] [Google Scholar]
37. Zheng CX, Wang SM, Bai YH, Luo TT, Wang JQ. Lentiviral Vectors and Adeno-Associated Virus Vectors: Useful Tools for Gene Transfer in Pain Research. 2018;301(5):825-36. [View at Publisher] [DOI] [PMID] [Google Scholar]
38. Jakobsson J, Lundberg C. Lentiviral vectors for use in the central nervous system. Molecular Therapy. 2006; 13(3): 484-93. [View at Publisher] [DOI] [PMID] [Google Scholar]
39. Azzouz M, Kingsman SM, Mazarakis ND. Lentiviral vectors for treating and modeling human CNS disorders. The Journal of Gene Medicine: A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications. 2004; 6(9): 951-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
40. Sano S, Wang Y, Evans MA, Yura Y, Sano M, Ogawa H, et al. Lentiviral CRISPR/Cas9-mediated genome editing for the study of hematopoietic cells in disease models. Journal of visualized experiments: JoVE. 2019; 152: 10.3791/59977. [View at Publisher] [DOI] [PMID] [Google Scholar]
41. Mátrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications. Molecular therapy. 2010;18(3):477-90. [View at Publisher] [DOI] [PMID] [Google Scholar]
42. Sinn P, Sauter S, McCray P. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors-design, biosafety, and production. Gene therapy. 2005;12(14):1089-98. [View at Publisher] [DOI] [PMID] [Google Scholar]
43. Yu YY, Xu MS, Liang H, Wang HY, Yu CQ, Liu Q. Review and future perspectives on the integration characteristics for equine lentivirus in the host genome. Pol J Vet Sci. 2023; 26(1): 163-72. [View at Publisher] [DOI] [PMID] [Google Scholar]
44. Bokhoven M, Stephen SL, Knight S, Gevers EF, Robinson IC, Takeuchi Y, et al. Insertional gene activation by lentiviral and gammaretroviral vectors. Journal of virology. 2009;83(1):283-94. [View at Publisher] [DOI] [PMID] [Google Scholar]
45. Ghosh S, Brown AM, Jenkins C, Campbell K. Viral vector systems for gene therapy: a comprehensive literature review of progress and biosafety challenges. Applied Biosafety. 2020; 25(1):7-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
46. Narayan O, Clements JE. Biology and pathogenesis of lentiviruses. Journal of General Virology. 1989;70(7):1617-39. [View at Publisher] [DOI] [PMID] [Google Scholar]
47. Kokkorakis N, Zouridakis M, Gaitanou M. Mirk/Dyrk1B Kinase Inhibitors in Targeted Cancer Therapy. Pharmaceutics. 2024;16(4): 528. [View at Publisher] [DOI] [PMID] [Google Scholar]
48. Becker W. A wake-up call to quiescent cancer cells - potential use of DYRK1B inhibitors in cancer therapy. Febs j. 2018;285(7):1203-11. [View at Publisher] [DOI] [PMID] [Google Scholar]
49. Gao J, Zhao Y, Lv Y, Chen Y, Wei B, Tian J, et al. Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells. Cancer Cell Int. 2013;13(1):2. [View at Publisher] [DOI] [PMID] [Google Scholar]
50. Peng S, Li W, Hou N, Huang N. A review of FoxO1-regulated metabolic diseases and related drug discoveries. Cells. 2020;9(1):184. [View at Publisher] [DOI] [PMID] [Google Scholar]
51. Mirshahi T, Murray MF, Carey DJ. The metabolic syndrome and DYRK1B. The New England journal of medicine. 2014;371(8):784-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
52. Mohamed YA, Hassaneen H, El-Dessouky MA, Safwat G, Hassan NA-M, Amr K. Study of DYRK1B gene expression and its association with metabolic syndrome in a small cohort of Egyptians. Molecular Biology Reports. 2021;48:5497-502. [View at Publisher] [DOI] [PMID] [Google Scholar]
53. Haldar SM. Metabolic syndrome: A family affair. Science Translational Medicine. 2014;6(239):239ec96-ec96. [View at Publisher] [DOI] [Google Scholar]
54. Singh R, Dhanyamraju PK, Lauth M. DYRK1B blocks canonical and promotes non-canonical Hedgehog signaling through activation of the mTOR/AKT pathway. Oncotarget. 2017;8(1):833. [View at Publisher] [DOI] [PMID] [Google Scholar]
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