Optimization of miR-22 expression cassette for rAAV delivery on diabetes

MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 were overexpressed in HEK293 and HeLa cells but the longer ones were more efficiently expressed. miR-22 may be placed in either introns or the 3′ UTR of a transgene for efficient overexpression. RNA polymerase III or II promoters were successfully utilized for miR expression but the latter showed higher expression levels in cell lines. Specifically, miR-22 was expressed efficiently together with an EGFP gene. After screening, a liver-specific TTR promoter was chosen to overexpress miR-22 in diabetic mice fed a high-fat diet. It was shown that miR-22 was overexpressed 2-3 folds which improved the insulin sensitivity significantly. The approach utilized in this study to optimize miR overexpression is a powerful tool for the creation of efficient rAAV vectors for the other miRs.

Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

Normal B Cell-Derived iPSCs Capable of Inducing RAS Mutants and Aid to Explore Myeloma-Initiating Cells

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain the potential to transform by reprograming, and the resulting chromosomal aberrations subsequently cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we previously established normal B cell-derived induced pluripotent stem cells (BiPSCs: BiPSC13 and MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and induce the expression of activation-induced cytidine deaminase (AID) by the Tet-off system (Sci Rep, 2017). Subsequently, we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5′-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14) carrying the DJ-rearranged allele of IgH that reciprocally translocated with CCND1 (G28). All these BiPSCs differentiated into hematopoietic progenitor cells (HPCs) (Sci Res, 2021). Next, we created AID fused with the hormone-binding domain of the estrogen receptor T2 mutant (AID-ER) to analyze the function of AID. The ER sequence of pCAG-ERT2CreERT2 (Addgene # 13777) was amplified by PCR and cloned into the downstream part of the TREtight promoter of pRetroX-Tight-Hyg as an open reading frame using an In-Fusion HD cloning kit. The ER sequence was also cloned into the same vector as a negative control for the analysis of AID function. These vectors were packaged into a retrovirus using gp293 and pMD2.G, and the viruses were infected into HEK293 cells expressing Tet-off transcription factor and fluorescence-deficient EGFP, in which the artificially-introduced stop codon was at the 106 thcodon of the EGFP gene. The expression of AID-ER fusion protein was induced by removal of doxycycline three days before analysis. AID activity was induced by the addition of 4-hydroxytamoxifen, an agonist of ER mutants, one day before analysis. Therefore, AID activity was monitored by detecting the frequency of EGFP-positive cells, i.e., the recovery of fluorescence, by flow-cytometry. Mutation of the artificial stop codon (from TAG to TAC; from stop codon to Tyrosine) was confirmed by sequencing the EGFP DNA amplified by PCR from the sorted EGFP-positive cells. Subsequently, we examined the introduction of mutated KRAS and NRAS genes into G28, because the most frequently occurring mutations in patients with MM are in KRAS and NRAS. Mutated KRAS (G12A) and NRAS (G13D) were amplified by RT-PCR from poly (A) mRNA of MM.1S and KMM1 of MM cell lines, respectively. After confirming the sequence of these mutants, the mutant cDNA of KRAS or NRAS was cloned into pRetroX-Tight-Hyg in combination with AID-ER. These two vectors were packaged into retroviruses, following which the viruses were infected into BiPSC13 with t(11;14) expressing Tet-off transcription factors, respectively. These BiPSCs also differentiated into HPCs. However, unlike cord blood, these HPCs did not differentiate into B lymphocytes by co-culture with BM stromal cell (MS-5). Therefore, further ingenuity is required to differentiate the BiPSC-derived HPCs into B lymphocytes. Disclosures No relevant conflicts of interest to declare.

Transgenesis biotechnological procedures influence on domestic duck embryos survival

Due to its high reproductive potential, short interval between generations and embryonic development outside the mother's body, the bird provides unique opportunities for its use in fundamental and applied biological research. The creation of a transgenic bird is complicated by the structure of its opaque egg cell with a large yolk and a unique reproductive system of this class. Direct microinjection of DNA into an oocyte, which is often used in mammals, is practically impossible for birds, since fertilization occurs in the infudibulum of the reproductive tract and can be polyspermic. Therefore, manipulations with the zygote turned out to be difficult for their use in creating a transgenic bird. Over the past decades, some alternative strategies have been developed for producing transgenic poultry using bizarre animals created by transferring blastodermal cells. However, to date, the efficiency of creating transgenic poultry in many cases remains very low, and the technique of using ducks to create transgenic poultry is practically not developed. Busulfan is used to suppress cell proliferation. Injection of busulfan into the pidembryonic cavity is one of the methods that increases the number of donor cells when creating chimeras. However, until now, methods of creating hermentative ducks chimeras face difficulties associated with the structure of the shell of waterfowl. Therefore, the aim of the work was to establish the effect of factors influencing the survival of transgenic embryos when using various methods of introducing a DNA construct into the duck genome. The objects of the study were ducks (Anas platyrhynchos) of the Shan partridge duck and Shaoxing breeds kept at the duck farm of Zhuji Guowei Poultry Development Co., Ltd, China. The studies were carried out in the poultry genetics laboratory of the Zhejiang Academy of Agricultural Sciences and on the duck farm of Zhejiang Generation Biological Science and Technology Co., Ltd. (Zhejiang Province, PRC). For the analysis of survival, we used embryos obtained by using various methods of introducing the DNA (insertion of the EGFP gene, mediated by homologous repair (HDR)) 1) direct injection of the DNA construct into the sub-embryonic cavity; 2) transfection of DNA with sperm; 3) injection of transfected donor blastomeres into recipient embryos after exposure to busulfan or ultraviolet radiation. A total more than 1100 eggs were examined. As a result of the direct injection of a transgenic DNA construction ( sub-embryonic cavity of 300 embryos, 35.7% of embryos did not develop after injection, 36% stopped developing at the time of the first ovoscopy (day 9 of incubation), 8% died within 10-15 days, 17, 3% - 16-25 days. In total, after direct injections, 9 live ducklings were received (the survival rate was 3%), of which 4 were transgenic. After insemination of ducks transfected with sperm, 292 eggs were laid for incubation. After the first ovoscopy, 51.4% of the eggs were unfertilized; 0.7% of embryos stopped developing at the time of the first ovoscopy (9 day of incubation), 1.0% died within 10-15 days, 17.8% - 16-25 days, 6.2% suffocated during hatching. In total, after using the transfected sperm, 67 live ducklings were obtained (the survival rate of embryos from fertilized eggs was 47.2%). Among 31 adult animals, 19 were transgenic. To sterilize recipient cells for the use of busulfan at a concentration of 300 ng per egg, followed by injection of blastodermal transfected donor cells, 200 embryos were examined, among which 61.0% of embryos developed after injection, 17.0% stopped in development at the time of the first ovoscopy (day 9 of incubation ), 12.5% of those died in the period of 10-15 days, 9.0% - 16-25 days. In total, after injections of busulfan at a concentration of 300 ng per egg, 1 live duckling was obtained (the survival rate was 0.5%). Using busulfan at a concentration of 150 ng per egg, 100 embryos were examined, among which 68.0% of embryos developed after injection, 11.0% stopped developing at the time of the first ovoscopy (day 9 of incubation), 5% died within 10-15 days, 14.0% - 16-25 days. In total, after injections of busulfan at a concentration of 150 ng per egg, 2 live ducklings were obtained (the survival rate was 0.5%). Using busulfan at a concentration of 75 ng per egg, 100 embryos were examined, among which 12.0% of embryos developed after injection, 27.0% stopped developing at the time of the first ovoscopy (9 day of incubation), 14.0% died in the period 10-15 days, 42.0% - 16-25 days. In total, after injections of busulfan at a concentration of 75 ng per egg, 5 live ducklings were obtained (the survival rate was 5%). Ultraviolet irradiation of 200 embryos for 1 hour followed by injection of blastodermal transfected donor cells resulted in death after injection of 20%, stopped developing 27.5% (9 days of incubation), 7.5% died within 10-15 days , 35.0% - 16-25 days. A total of 20 live ducklings were obtained using ultraviolet radiation (survival rate was 10%). Among 13 adult animals gave offspring, 7 were transgenic chimeras. The use of ultraviolet light has reduced the impact of egg infection due to the structure of waterfowl shells. Thus, the safest for the survival of embryos was the method of insemination of ducks with transfected sperm, using which 47.2% of embryos survived.

Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3’ truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5’ truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.

144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

Torovirus (ToV) has recently been classified in the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. Reverse genetics systems for many CoVs have been established, but none exist for ToVs. Here, we describe a reverse genetics system using a full-length infectious cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV containing genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the Hemagglutinin-esterase (HE) gene was manipulated, since cell-adapted wtBToV generally loses the full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued; these showed no significant differences in cell growth, suggesting that HE is not essential for viral growth in cells. Then, recombinant virus in which HE was replaced by the Enhanced Green Fluorescent Protein (EGFP) gene expressed EGFP in infected cells, but showed significantly reduced viral growth compared to wtBToV. Moreover, the recombinant virus readily deleted the EGFP gene after one passage. Interestingly, one variant with mutations in non-structural proteins (NSPs) showed improved EGFP expression and viral growth during serial passages, although it eventually deleted the EGFP gene, suggesting that these mutations contributed to EGFP gene acceptance. These recombinant viruses provide new insights regarding BToV and its reverse genetics will help advance understanding of this neglected pathogen.ImportanceToVs are diarrhea-causing pathogens that have been detected in many species, including humans. BToV has spread worldwide, leading to economic losses. We developed the first reverse genetics system for Tobaniviridae using a BAC-based BToV. Using this system, we showed that recombinant BToVs with HEf and HEs showed no significant differences in cell growth. In contrast, clinical BToVs generally lose the HE gene after a few passages but some recombinant viruses retained the HE gene for up to 20 passages, suggesting some benefits of HE retention. The EGFP gene of the recombinant viruses was unstable and was rapidly deleted, likely via negative selection. Interestingly, one virus variant with mutations in NSPs was more stable, resulting in improved EGFP-expression and viral growth, suggesting that the mutations contributed to some acceptance of the exogenous EGFP gene without clear positive selection. The recombinant BToVs and reverse genetics developed here are powerful tools for understanding fundamental viral processes and their pathogenesis and for developing BToV vaccines.

Designing of a DNA matrix for transgene integration into the bovine beta-lactoglobulin gene locus using CRISPR/Cas9 technology

Beta-lactoglobulin (BLG) is the main protein in milk serum in almost all mammals, with the exception of rodents and primates. Regulatory regions of the beta-lactoglobulin gene in ruminants (sheep, goats, and cattle) as part of genetic constructs provide tissue - specific expression of recombinant protein in the mammary gland and have been actively used in genetic engineering since the beginning of the era of creating transgenic animals. To work effectively with the CRISPR/Cas9 genomic editing method, it is necessary to know the exact DNA sequence of the target gene: this is necessary both for creating a DNA matrix for homologous recombination and for the targeted accuracy of guide RNAs. A polymorphic variant of the bovine BLG gene was identified, whose sperm was used to fertilize cow oocytes in vitro. The aim of this work was to create a plasmid containing 5’ - and 3’ - arms of homology (ha) to the bovine BLG gene. Based on ??TZ57R/T, the pTZhaBLG plasmid was obtained, which has a unique site for EagI restriction at the junction of the homology arms. A fragment containing a biologically active protein gene can be embedded in the resulting plasmid at this restriction site. We created the pBLGcmvEGFP plasmid containing the green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter: protein expression can serve as a reliable indicator of successful integration of the transgene into the genome. The resulting plasmids in circular or linearized form are intended for site-specific integration by homologous recombination repair into the BLG gene using CRISPR/Cas9 components.

Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.

Generation of a bacterial clone for assessing the impact of climatic stress conditions on microbial proliferation

Documented increases in atmospheric Carbon Dioxide (CO2) concentrations have contributed to a rise in average global temperatures. Environmental variation due to climate change is expected to affect the growth of microorganisms. Hence, there is a need to assess the induced adaptations of microorganisms, which are common biological contaminants, to environmental changes. Therefore, an enhanced green fluorescent protein (eGFP) expressing Escherichia coli BL21(DE3) clone was generated. Plasmid pAP1698-4 was used as the donor for the eGFP gene and pD454-MBP as the recipient plasmid to produce pD454-MBPeGFP. Expression of eGFP in the clone was confirmed using confocal microscopy. The growth of the clone was characterised by plate counting technique. Variation in the length of the lag phase, λ, and growth rate, μmax, kinetic parameters of the clone was observed, compared to the wildtype BL21(DE3). A live/dead kinetic assay, using eGFP for the quantification of live cells and propidium iodide (PI) as a stain for dead cells, was optimised using a microplate reader with controlled temperature and CO2 conditions. Full growth curves were collected when culture media was inoculated with 4 to 6 Log10CFU.mL-1. The optimal PI concentration was 150 nM; higher concentrations inhibited growth, and lower concentrations gave no signal difference compared to the blank. The growth kinetics of the clone under different environmental conditions; between 400 ppm to 2500 ppm CO2, combined with 37°C to 42°C, were evaluated using the live/dead kinetic assay, allowing assessment of response to induced environmental stress.