Vector Backbone Research Articles

Improving transient expression in N. benthamiana by suppression of the Nb-SABP2 and Nb-COI1 plant defence response related genes.

Currently transient expression is one of the preferred plant-based technologies for recombinant protein manufacturing, particularly in respect to pharmaceutically relevant products. Modern hybrid transient expression systems combine the features of Agrobacterium tumefaciens and viral vectors. However, host plant reaction to Agrobacterium-mediated delivery of gene of interest can negatively affect foreign protein accumulation. In this study, we investigated whether the modulation of plant immune response through knockdown of the Nb-SABP2 and Nb-COI1 N. benthamiana genes could improve recombinant protein yield. In plants, the SABP2 and COI1 proteins are involved in the salicylic acid and jasmonic acid metabolism, respectively. We exemplified the utility of this approach with the green fluorescence (GFP) and β nerve growth factor (βNGF) proteins: compared to the tobacco mosaic virus (TMV)-based vector the Nb-SABP2 and Nb-COI1-suppressed plants provided an increased recombinant protein accumulation. We also show that this strategy is extendable to the expression systems utilizing potato virus X (PVX) as the vector backbone: the enhanced amounts of βNGF were detected in the Nb-SABP2 and Nb-COI1-depleted leaves co-infiltrated with the PVX-βNGF. These findings suggest that modulating host plant reaction to agrodelivery of expression vectors could be useful for improving transient foreign protein production in N. benthamiana.

Recombinant Marek’s disease virus type 1 provides full protection against H9N2 influenza A virus in chickens

The H9N2 subtype of the avian influenza virus (AIV) poses a significant threat to the poultry industry and human health. Recombinant vaccines are the preferred method of controlling H9N2 AIV, and Marek’s disease virus (MDV) is the ideal vector for recombinant vaccines. During this study, we constructed two recombinant MDV type 1 strains that carry the hemagglutinin (HA) gene of AIV to provide dual protection against both AIV and MDV. To assess the effects of different MDV insertion sites on the protective efficacy of H9N2 AIV, the HA gene of H9N2 AIV was inserted in UL41 and US2 of the MDV type 1 vector backbone to obtain recombinant viruses rMDV-UL41/HA and rMDV-US2/HA, respectively. An indirect immunofluorescence assay showed sustained expression of HA protein in both recombinant viruses. Additionally, the insertion of the HA gene in UL41 and US2 did not affect MDV replication in cell cultures. After immunization of specific pathogen-free chickens, although both the rMDV-UL41/HA and rMDV-US2/HA groups exhibited similar levels of hemagglutination inhibition antibody titers, only the rMDV-UL41/HA group provided complete protection against the H9N2 AIV challenge, and also offered complete protection against challenge with MDV. These results demonstrated that rMDV-UL41/HA could be used as a promising bivalent vaccine strain against both H9N2 avian influenza and Marek’s disease in chickens.

Construction and Stability of All-in-One Adenovirus Vectors Simultaneously Expressing Four and Eight Multiplex Guide RNAs and Cas9 Nickase.

CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

Effect of Prior ChAdOx1 COVID-19 Immunisation on T-Cell Responses to ChAdOx1-HBV.

There are varying data concerning the effect of prior anti-vector immunity on the T-cell response induced by immunisation with an identical vectored vaccine containing a heterologous antigen insert. To determine whether prior exposure to ChAdOx1-SARS-CoV2 immunisation (Vaxzevria®) impacts magnitudes of antigen-specific T-cell responses elicited by subsequent administration of the same viral vector (encoding HBV antigens, ChAdOx1-HBV), healthy volunteers that had received Vaxzevria® (n = 15) or the Pfizer or Moderna mRNA COVID-19 vaccine (n = 11) between 10 and 18 weeks prior were recruited to receive a single intramuscular injection of ChAdOx1-HBV. Anti-ChAdOx1-neutralising antibody titers were determined, and vector or insert-specific T-cell responses were measured by a gamma-interferon ELISpot and intracellular cytokine staining (ICS) assay using multiparameter flow cytometry. Participants were followed for three months after the ChAdOx1-HBV injection, which was well-tolerated, and no dropouts occurred. The baseline ChAdOx1 neutralisation titers were higher in the Vaxzevria® cohort (median of 848) than in the mRNA cohort (median of 25). T-cell responses to HBV antigens, measured by ELISpot, were higher on day 28 in the mRNA group (p = 0.013) but were similar between groups on day 84 (p = 0.441). By ICS, these differences persisted at the last time point. There was no clear correlation between the baseline responses to the adenoviral hexon and the subsequent ELISpot responses. As vaccination within 3 months using the same viral vector backbone affected the insert-specific T-cell responses, a greater interval after prior adenoviral immunisation using heterologous antigens may be warranted in settings in which these cells play critical roles.

Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT

BackgroundTransgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called “pronuclear injection-based targeted transgenesis” (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad.ResultsTo enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice.ConclusionsSimultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Quantification of Lymphangiogenesis in the Murine Lymphedema Tail Model Using Intravital Microscopy.

Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated five million Americans. There is no cure for this disease. Assessing lymphatic growth is essential in developing novel therapeutics. Intravital microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous nonviral vector gene delivery using a chip with nanochannel poration in a rapid (<100 ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics in the live mice model. Methods and Results: The murine tail model of lymphedema was utilized. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: TNTpCMV6 group receives pCMV6 (expression vector backbone alone) (n = 6); TNTProx1 group receives pCMV6-Prox1 (n = 6). Lymphatic vessels (fluorescein isothiocyanate [FITC]-dextran stained) and lymphatic branch points (indicating lymphangiogenesis) were analyzed with the confocal/multiphoton microscope. The experimental group TNTProx1 exhibited reduced postsurgical tail lymphedema and increased lymphatic distribution compared to TNTpCMV6 group. More lymphatic branching points (>3-fold) were observed at the TNT site in TNTProx1 group. Conclusions: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. IVM can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations.

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

Specific antibody production using recombinant proteins to elucidate seed transmission and nuclear localization of Coguvirus citrulli and Coguvirus henanense in radicles of watermelon crop

Watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, both belonging to the genus Coguvirus (family Phenuiviridae), have been identified in watermelon plants in Brazil. To study tissue tropism and the potential for seed transmission of these viruses, we initially planned to produce specific antibodies. However, difficulties in isolating and propagating the virus in host plants hindered the purified virus preparations. To overcome this problem, the nucleocapsid (N) proteins of WCLaV-1 and −2 were produced using the pepper ringspot virus vector. The N protein genes and the vector backbone were prepared by (RT-)PCR and ligated by Gibson assembly. The constructs were agro-infiltrated in Nicotiana benthamiana plants. The expressed N proteins were purified and used for polyclonal antibody production. The specificity of both antibodies was confirmed by antigen-coating ELISA, tissue-blot immunobinding assay and Western blot. By antigen-coating ELISA demonstrated that WCLaV-1 showed 93.1% of seed-transmission, while WCLaV-2 showed only 17.8%. The N protein of WCLaV-1 was detected in the cytoplasm of the seed tissues. It was also found in the nuclei of the radicle, as confirmed by confocal microscopy. We concluded that the antibodies exhibited both a high titer and sufficient specificity for use in ELISA-based diagnostics and for subcellular localization study.

Topical tissue nanotransfection of Prox1 is effective in the prophylactic management of lymphedema

Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. There is no cure for the disease. Clinically, a preventative surgical approach termed immediate lymphatic reconstruction (ILR) has gained traction. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection (TNT) technology utilizes a direct, transcutaneous non-viral vector, gene delivery using a chip with nanochannel poration in response to a rapid (<100ms) focused electric field. The purpose of this study is to experimentally prevent lymphedema using focal delivery of a specific gene Prox1 (a master regulator of lymphangiogenesis). TNT was applied to the previously optimized lymphedematous mice tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: Group I (sham) was given pCMV6 (expression vector backbone alone) and Group II was treated with pCMV6-Prox1. Group II mice had decreased tail volume (47.8%) compared to sham and greater lymphatic clearance on lymphangiography. Immunohistochemistry showed greater lymphatic vessel density and RNA sequencing exhibited reduced inflammatory markers in Group II compared to Group I. Prox1 prophylactically delivered using TNT to the surgical site on the day of injury decreased the manifestations of lymphedema in the murine tail model compared to control.

YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes.

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.

Down-regulation of key regulatory factors in sphingosine-1-phosphate (S1P) pathway in human lung fibroblasts transfected with selected microRNAs.

Sphingosine 1 phosphate (S1P) is involved in the pathogenesis of asthma by stimulation of the alpha-smooth muscle actin (SMA) expression and remodeling of fibroblasts. This study was designed to determine the effects of selected micro RNAs in regulation of S1P and related metabolic pathways in a human lung fibroblast cell line. The fibroblast cell line (CIRC-HLF, C580) was cultured and transfected with individual viral vectors carrying miR124, mi125b, mi133b or mi130a. After 48 hours, expression level of miRNAs and their target genes, sphingosine kinase 1(SPHK1), sphingosine 1-phosphate lyase 1 (SGPL1), sphingosine 1- phosphate receptor 1 (S1PR1) and sphingosine 1- phosphate receptor 2 (S1PR2) were determined. Expression of miRNA and mRNA determined by reverse transcriptionquantitative polymerase chain reaction (qPCR) showed that the expression level of the miRNAs was significantly higher in human lung fibroblasts following transfection compared to controls (vector backbone without miRNA). The expressions of miRNAs-targeted genes were significantly downregulated in transfected fibroblasts compared to control cells (p<0.05). Data show that miR 124, miR 125b, miR 133b and miR130a by targeting regulatory genes in S1P-pathway can down-regulate key factors such as SPHK1, SGPL1, S1PR1 and S1PR2 genes in lung fibroblasts. The results showed that S1P pathway and key factors are suppressed in lung fibroblasts expressing miR124, miR125b, miR130a or miR133b. It appears that suppression of any of the intermediate factors in S1P by miRNA can affect the regulation of the entire S1P pathway.

Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction.

Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one. We present here a simple method that requires only crude oligos, commercially available high-fidelity enzymes, and the success rate is close to 100%. In addition, up to 6 different mutations can be introduced in one reaction without causing any fortuitous change in the vector backbone. Using this strategy, we have introduced 32S/T➔A substitutions in the N-terminus head and 13 changes in the C-terminus tail domain of vimentin.

The application of gene-editing technologies in plants

In recent years, we have witnessed a spurt of progress in modern molecular biology, thus enabling the perfection of genome sequencing. As a result, research into gene function has gained popularity among scientists. Since gene editing has served as an essential tool for the exploration of plant gene function, the utilization of genome editing technology contributes most to the genetic upgrading and crop breeding through modification on plant genome. Typical cases where CRISPR/Cas system has been applied to model species including Arabidopsis thaliana, Nicotiana tabacum and Oryza sativa to alleviate salinity stress and figure out the gene behind traits relative to their architecture and yield. The modified CRISPR/Cas9 system requires only simple cloning compared with the other two types of gene editing tools and the design of target sequences is easy, the construction of backbone vectors is simple, and the cost is relatively low. The CRISPR/Cas9 system, which was awarded the 2020 Nobel Prize in Chemistry, has transformed the molecular life sciences by enabling researchers to accurately modify the DNA of animals, plants, and microorganisms. This review thoroughly introduced gene editing technologies currently applied in plants, including map cloning, RNAi technology and targeted genome editing technology, containing ZFNs, TALEN, CRISPR/Cas9. The review revealed the difficulties and potential uses of genome editing technology in plants while concentrating on its theory and viability.

An Adapted GeneSwitch Toolkit for Comparable Cellular and Animal Models: A Proof of Concept in Modeling Charcot-Marie-Tooth Neuropathy.

Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch™ technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch™ cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical "teardrop" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.

TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method.

Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5U of T5 exonuclease at 0°C for 5min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.

153. Quantification of Lymphangiogenesis in Murine Lymphedema Tail Model Using Intravital Microscopy

PURPOSE: Lymphedema is limb swelling caused by lymphatic dysfunction. It occurs in 30% of patients that undergo axillary lymph node dissection in the treatment of breast cancer. There is no cure for this disease. Understanding the mechanisms of lymphatic growth will play a pivotal role in developing therapeutic strategies against these conditions. Visualization of lymphangiogenesis and functional assessment remains a challenge. Intravital two-photon microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous non-viral vector gene delivery using a chip with nanochannel poration in a rapid (<100ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics. METHODS: The murine tail model of lymphedema was utilized. A 3 mm full thickness skin excision and lymphatic vessel disruption was performed 20 mm from the base of the tail in twelve C57BL/6 mice. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: Group I (control) was given pCMV6 (expression vector backbone alone) (n=6); Group II had pCMV6-Prox1 (n=6). Post-TNT (day 10), a 3 cm segment of murine tail was deskinned distal to the site of occlusion to optimize visualization. FITC-Dextran (2000 kD) injected intradermally at the distal tail region for lymphatic uptake. Lymphatic vessels are visualized at the second skin excision site with the Leica SP8 Confocal/Multiphoton Microscope and assessed for number of branching points to determine the newly formed lymphatics. Lymphatic vessel density was also observed by immunostaining with anti-Podoplanin antibody. RESULTS:The experimental group II exhibited increased branching points (3-fold) using filamentation analysis compared to control group I at the site of TNT treatment (n=6, p<0.05). Increased lymphatic vessel density was also observed with Podoplanin immunostaining post-TNT application. Intensity quantification of immunohistochemistry revealed greater expression of Podoplanin in Group II when compared to Group I (n=6, p<0.05). CONCLUSION:This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. Intravital microscopy can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator

Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.