Murine Leukemia Virus Reverse Transcriptase Research Articles

Development of Moloney Murine Leukemia Virus Reverse Transcriptase Fused with Archaeal DNA-binding Protein Sis7a.

Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has its shortcomings, especially in terms of processivity and thermostability. According to a previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has been proven to enhance its performance. Furthermore, recent studies have also stated that the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. As an early stage of enzyme development, this study aimed to design, express, and purify enzymatically active MMLV RT fused with archaeal DNA-binding protein. RT fusion proteins were designed and evaluated using in silico methods. The RT fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse transcriptional activity was proved using reverse transcription quantitative polymerase chain reaction (RT-qPCR). This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT fusion was successfully expressed and purified. It was also known that the optimal condition for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that the RT fusion has the reverse transcriptional activity. This study shows that the designed MMLV RT Sis7a fusion can be expressed and purified, is enzymatically active, and has the potential to be developed as an improved RT enzyme. Further study is still needed to prove its thermostability and processivity, and further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial use.

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, invivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

An optimized prime editing system for efficient modification of the pig genome.

Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

Crystal structures of 'ALternative Isoinformational ENgineered' DNA in B-form.

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host-guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5'-CTTBPPBBSSZZSAAG, 5'-CTTSSPBZPSZBBAAG and 5'-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, and B:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters of P:Z and B:S pairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further, Z can participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

FP.42 Correction of point mutations in the DMD gene using the prime editing

Duchenne muscular dystrophy (DMD) is a X-linked hereditary disease responsible for serious burden, pain and damages to patients and their families. About 20 000 children new cases are diagnosed worldwide each year. Existing experimental treatments with antisense oligonucleotides or aminoglycosides provide limited phenotypic improvement. Our study aimed to experiment the CRISPR-Cas9 PRIME editing technology as therapeutic approach. This technology uses a PRIME editor plasmid (PE2) coding for a Moloney murine leukemia virus reverse transcriptase fused with the Cas9 nickase, and a plasmid coding for a prime editing guide RNA (pegRNA) containing in addition to a sgRNA, a primer binding site (PBS) and a reverse transcriptase template (RTT). This system permits specific nucleotide substitutions, deletions or insertions in the genome. We first designed different pegRNAs targeting several human DMD exons (6, 9, 20, 35, 43, 52, 55, 59 and 61) responsible for DMD in Canadian population to introduce a STOP codon by modifying a single nucleotide. We next designed other pegRNAs for the correction of c.428 G>A and c.8713 C>T point mutations respectively in DMD exon 6 and exon 59 in patients' human myoblast. HEK293T and myoblast cells were harvested three to five days post treatment with PE2 and pegRNA plasmids. Parts of myoblasts were transformed into myotubes to obtain proteins for western blot analysis. Fragment of targeted exons were PCR amplified and sequenced by Sanger and Illumina deep sequencing method. Results were analysed using the EditR and CRISPRESSO programs to estimate the editing percentage. We confirmed that PRIME editing permitted the specific C˃T, A˃G, G˃C, G˃A and G˃T substitutions in the DMD gene with an editing efficiency between 3 to 9% and 6 to 21% respectively using PE2 and PE3 approach in HEK293T cells creating point mutations. An additional mutation in the PAM sequence permitted to achieve up to 40% modification. For the correction of point mutations in patients' myoblasts, the modification of RTT length in combination to synonymous additional mutations around the target mutation showed up to 23% correction for a single electroporation with dystrophin expression detected by western blot. Thus Prime editing permits the correction of point mutations in the DMD gene.

Engineered CRISPR prime editors with compact, untethered reverse transcriptases.

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L−1 to 0.175 g L−1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10930-022-10066-5.

First Report of Chinese Wheat Mosaic Virus that Infects Barley in Japan.

Chinese wheat mosaic virus (CWMV), a member of the genus Furovirus in the family Virgaviridae (Adams et al. 2017), has a positive-sense RNA genome and is transmitted by Polymyxa graminis. CWMV is a causal agent of yellow mosaic disease in winter wheat in China (Guo et al. 2019). CWMV has also been detected in wheat plants in limited areas of the northern Japan (Nagano and Iwate Prefectures) (Fuji et al. 2022; Maeshima et al. 2010; Shirako and Maejima 2008). In preliminary tests using Western blotting with an antiserum raised against CWMV capsid protein (by Dr Y. Shirako, Tokyo University), we detected a furovirus in a breeding line of barley, “Tozan Kawa 111” (Hordeum vulgare L.) (collected in April 2012), grown in an experimental field infested with CWMV and wheat yellow mosaic virus (genus Bymovirus) in Nagano Prefecture. To investigate the infection of barley plants with cereal plant-associated soil-borne viruses, we collected leaf samples of “Tozan Kawa 111” plants (ten plants) showing yellow mosaic symptoms, but with not apparent wilting or stunting (Fig. S1A) in April 2016 (2015/16 growing season). Total RNA was extracted from symptomatic leave samples with TaKaRa RNAiso reagent (TaKaRa Bio) and subjected to reverse transcription polymerase chain reaction (RT-PCR) to detect virus agents. After cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific) with random hexamers, PCR amplification with QuickTaq HS Dye Mix (Toyobo Co.) was conducted using primer sets specific to two furoviruses and three bymoviruses (Fig. S1B) known to infect wheat and/or barley plants in Japan (Tamada and Kondo 2013). RT-PCR analysis detected infection with CWMV in the leaf samples of “Tozan Kawa 111” plants (Fig. S1B), but not the other soil-borne viruses tested. The amplified PCR products (752 and 718 bp for CWMV RNA1 and RNA2, respectively) were purified by Wizard® SV Gel and PCR Clean-Up System (Promega) and subjected to Sanger sequencing to confirm their nucleotide sequences. The virus sequences from PCR amplicons were deposited in GenBank/DDBJ/ENA with accession numbers LC657081 (RNA1) and LC657082 (RNA2). Nucleotide Basic Local Alignment Search Tool (BLASTn) analysis showed that the sequences have 98.7% and 98.8% nucleotide sequence identity with RNA1 of CWMV Japanese northern isolate (accession No, AB299271) and RNA2 of CWMV Nagano-A isolate (AB935554), respectively. Identical sequences were also found in symptomatic wheat leaf samples (“Fukuho Komugi” cultivar) obtained from the same field (Fig. S1B). Rod-shaped particles were observed by transmission electron microscopy (Hitachi H-7650) in symptomatic “Tozan Kawa 111” leaf samples (Fig. S1C). Using reverse transcription loop-mediated isothermal amplification (Fukuta et al. 2013), CWMV was detected in “Tozan Kawa 111” plants in the same field in the 2015/16–2017/18 growing seasons, but not in the 2018/19–2020/21 growing seasons (Fig. S1D). In the 2015/16–2017/18 growing seasons, CWMV was detected in the barley plants (pooled five plant samples) cultivar “Kashima-mugi”, which showed similar yellow mosaic symptoms (Fig. S1D), but not in most of the other barley variants planted in the field. To our knowledge, this is the first report of CWMV field infection in plants other than wheat (Kuhne 2009). Further extensive virus screening in the fields and virus inoculation experiments are necessary to understand the pathology of CWMV in barley and possibly in other cereal crops.

Polypyridyl ruthenium complexes as bifunctional TAR RNA binders and HIV-1 reverse transcriptase inhibitors

Inhibitors of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase are central to anti-HIV therapy. Most of their targets are enzymes, while very few could bind to viral RNA. Here we designed four new polypyridyl Ru(II) complexes, which could bind HIV-1 TAR RNA tightly and selectively by molecular recognition of hydrogen bonds, further stabilize the Ru(II)-RNA bound system by electrostatic attraction, and efficiently inhibit the Moloney murine leukemia virus (M-MuLV) and HIV-1 reverse transcriptase. The polypyridyl Ru(II) complexes also have physical and chemical advantages, including high chemical stability and photostability, sensitive spectroscopic responses to HIV TAR RNA, and low toxicity to normal cells. This work also provides valuable drug design strategies for acquired immune deficiency syndrome (AIDS) and other reverse transcriptase related disease research, such as hepatitis C virus (HCV), Ebola virus (EBOV), influenza A virus, and most recently the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

First Report of Tomato Brown Rugose Fruit Virus on Greenhouse Tomato in Syria.

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower's cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer's instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer's instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5'-CATATCTCTCGACACCAGTAAAAGGACCCG-3') and ToBRFV-R2 (5'-TCCGAGTATAGGAAGACTCTGGTTGGTC-3') targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

Homebrew reagents for low-cost RT-LAMP.

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has gained popularity for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The high specificity, sensitivity, simple protocols, and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents, and their distribution under cold chain shipping limit RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits, and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range, and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs; thus, we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low-cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Genome-wide specificity of prime editors in plants.

Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome.

Constructing a DNAzyme Delivery and Expression System for Escherichia coli: A Prototype for Phage Therapy

Antisense oligonucleotides exhibit high potential for use as therapeutic agents. '10-23' DNAzymes are antisense molecules with a high chemical stability and catalytic efficiency. In the present study, we developed a phagemid containing a DNAzyme expression system regulated by two promoters. One of these promoters, pA1, promotes constitutive expression of Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT). The other promoter, plac, regulates transcription of the RNA substrate from which MoMuLV-RT produces the DNAzyme by reverse transcription. The ftsZ DNAzyme was used to validate this expression system in the phagemid, named pDESCP. ftsZ DNAzyme expression altered the morphological pattern of Escherichia coli from a bacillary to filamentous form. In E. coli FtsZ is the primary component of the cell division apparatus, forming a structure known as Z-ring, which is the place of division. It is suggested that the DNAzyme ftsZ is decreasing the translation of this protein. Delivery of pDESCP into F+ strain of E. coli cells, using VCSM13, and the possible insertion of other DNAzymes into the cassette makes this phagemid an important prototype for phage therapy.

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.

Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region

Abstract Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) is widely used in research and clinical diagnosis. Improvement of MMLV RT thermostability has been an important topic of research for increasing the efficiency of cDNA synthesis. In this study, we attempted to increase MMLV RT thermostability by introducing a disulfide bridge in its RNase H region using site-directed mutagenesis. Five variants were designed, focusing on the distance between the two residues to be mutated into cysteine. The variants were expressed in Escherichia coli and purified. A551C/T662C was determined to be the most thermostable variant.

Genotyping and prevalence of hepatitis c virus in dialysis patients in Gandhi general hospital - A tertiary care centre

Context: The present study was undertaken to determine the genotyping and prevalence of hepatitis c virus in dialysis patients as HCV genotyping has become a part of the pre-treatment evaluation. Studies correlating risk factors and genotypes of HCV infection prevalent in Haemodialysis patients are scanty. Therefore our study is conducted to study the prevalence, genotypes and risk factors in order to detect and try to curb HCV infection. Aims: To study the genotypes of hepatitis C infection and its prevalence in hemodialysis patients. Settings and Design and Period: Study was conducted in Gandhi hospital Secunderabad in 1 year period as Cross-sectional study. Materials and Methods: A total of 225 serum samples were collected from chronic renal failure patients who were undergoing Haemodialysis over a period of 1 year. The samples were screened for anti HCV antibodies by third generation ELISA test. 25 anti-HCV positive samples and 40 anti-HCV negative samples were randomly selected for HCV RNA detection using Trizol-Chloroform-Isopropyl alcohol method, HCV RNA was reverse transcribed with 200 U of maloney murine leukemia virus reverse transcriptase and random hexamers. Nested PCR was done. HCV RNA positive samples were genotyped with primers specific for core region of different HCV types by type specific PCR. Statistical Analysis Used: Statistical analysis of the data was done by chi-square (c2) test using EPIINFO 2000 software. The values of chi square test are interpreted as not significant >0.05, highly significant < 0.001. Results: Out of 225 hemodialysis patients 38 (16.8%) patients were anti HCV positive. Duration of dialysis was significantly longer in anti-HCV antibodies positive group with dialysis duration more than 2 years. Seropositivity is more in HD patients having dialysis at more than one centre. HCV RNA was detected in randomly selected 13/25 (52%) anti HCV positive patients and in 5/40 (12.5%) anti HCV negative patients. The genotype distribution

One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.

LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 6 PRIME editing permits to introduce specific point mutations in the gene coding for dystrophin.

Duchenne Muscular Dystrophy is a X-linked hereditary disease with the prevalence of 19.8 per 100 000 males’ birth caused by mutations in the DMD gene coding for dystrophin. Currently available clinical therapies with corticosteroids or with morpholino antisense oligomer injections provides limited phenotypic improvement. Our study aimed to experiment the PRIME editing technology. This technology uses a PRIME editor plasmid (PE2) coding for a Moloney murine leukemia virus reverse transcriptase fused with the Cas9 H840A nickase, and a plasmid coding for a pegRNA containing a primer binding sites (PBS) and a reverse transcriptase template (RTT). It permits specific nucleotide substitutions, deletions or insertions in the genome. We designed different pegRNAs targeting several hDMD exons (9, 20, 35 and 61) to introduce a STOP codon by modifying a single nucleotide. HEK293T cells were harvested from DMEM culture media three days after being simultaneously transfected with the PE2 and pegRNA plasmids. Parts of the targeted exons were PCR amplified and sequenced with the Sanger method. Results were analysed using the EditR program to estimate the editing percentage. We confirmed that PRIME editing permits the specific C to T and G to T substitutions in the DMD gene with an editing efficiency between 6 to 9%. Repeated transfections 6 days after the first one showed up to 15 % edition in exons 9 and 35. Thus PRIME editing permits the specific substitutions in DMD gene and might be used to correct point mutations in the DMD gene to lead to dystrophin expression.

Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system

Abstract Reverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that C- terminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase.

190 Effects of endocrine-disrupting chemical on calcium signalling in cardiomyocyte differentiation from mouse embryonic stem cells

Endocrine-disrupting chemicals (EDC) are substances similar to steroid hormones that can disturb normal physiological functions of male and female reproductive organs. Endocrine-disrupting chemicals tend to bind to steroid hormone receptors. Sex steroid hormones modulate calcium signalling in the cardiac muscle in early embryo development. Among the steroid hormones, progesterone (P4) has been reported to affect blood pressure and other aspects of the cardiovascular system. The EDC that have similar structure to P4, such as octyl-phenol (OP) and bisphenol A (BPA), are potentially harmful to development of the heart. To confirm the effect of OP and BPA on early differentiation of mouse embryonic stem cells (mES) into cardiomyocytes, the hanging-drop method with mESC cell line (ES-E14TG2a) was used for forming embryoid bodies. Pluripotent mESC were cultured in basal medium with leukemia inhibitory factor and grown on mitomycin C-treated mouse embryonic fibroblasts in a 60-mm plate at 37°C in a 5% CO2 humidified tissue culture incubator with basal medium consisting of DMEM/F-12 supplemented with nonessential amino acids, 10% heat-inactivated and certified fetal bovine serum (FBS), 2-mercaptoethanol, penicillin, and streptomycin. The mouse embryoid bodies (mEB) were suspended onto 6-well plates and cultured with differentiation medium containing steroid-free FBS without leukemia inhibitory factor. Progesterone, OP, and BPA were added on Day 2 from mEB attachment. In addition, mifepristone (RU486), an antagonist for progesterone receptor (PR), was used to confirm the effect of P4 through PR. To determine whether RU486 is capable of attenuating the inhibition effect of P4, RU486 was added for 1 day starting on Day 11. Assessment of cardiomyocyte differentiation was determined by checking the number of beating cell populations divided by the total number of attached mEB. Total RNA was extracted using Trizol reagent and synthesised to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase. Messenger RNA level was assessed using quantitative real-time PCR. To investigate calcium signalling, the mRNA levels of calcium channel gene Trpv2 and contraction-related genes Ryr2, Cam2, and Mylk3 were analysed. Beating ratio was decreased in P4, OP, and BPA treatments. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison tests, and P&amp;lt;0.05 was considered statistically significant in least 3 different replicates. The mRNA level of Pgr was significantly increased in P4, OP, and BPA-treated group. However, the mRNA level of calcium channel gene Trpv2 was significantly decreased in the P4, OP, and BPA-treated group. Expression of contraction-related genes such as Ryr2, Cam2, and Mlck3 were significantly decreased in the P4, OP, and BPA-treated group. In addition, treatment with RU486 rescues altered calcium channel gene and contraction-related genes. Taken together, these results suggest that OP and BPA may affect the differentiation of mESC into cardiomyocyte and disrupts differentiation of cardiomyocytes.