Multiple Cloning Site Research Articles

Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly

BackgroundThe ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes.ResultsWe developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated.ConclusionsOur protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)orphytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

Suicide gene therapy-mediated purine nucleoside phosphorylase/fludarabine system for in vitro breast cancer model with emphasis on evaluation of vascular endothelial growth factor promoter efficacy.

In this study, a suicide gene therapy approach was optimized by a non-viral polyplex system based on pEGFP-N1 vector harboring purine nucleoside phosphorylase gene conducted by vascular endothelial growth factor promoter for an in vitro breast cancer model (4T1 cell line). The VEGF promoter and purine nucleoside phosphorylase gene were cloned into the vector from the source of 4T1 and E. coli genomic DNA, respectively. A gene construct was developed by replacing VEGF promoter instead of CMV promoter in pEGFP-N1vector. PNP gene was integrated in to the multiple cloning site of the obtained vector. On the other hand, a construct from pEGFP-N1 harboring PNP gene under the control of the original CMV promoter was developed. The transfection method using cationic polymer was optimized based on N/P ratio, cell cytotoxicity, polyplex size, zeta potential and the green fluorescent protein (GFP) expression by fluorescent microscopy and flowcytometry. Also, the effect of hypoxia condition induced by 0.5mM H2O2 on the promoter efficiency was investigated. The results showed that the performed gene delivery system is capable of the gene transfection to more than 30% of the cancer cells with both VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1 plasmids. The hypoxia condition did not show a significant effect on the VEGF promoter. But, it revealed that bystander effect can improve the efficacy of this system and reduce drug IC50 to 2 and fourfold for plasmids VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1, respectively. These results showed that the bystander effect could almost compensate the low efficiency of non-viral gene delivery systems. We suggest that the tumor-specific gene expression system mediated by the VEGF promoter can be especially useful in the present model of breast cancer gene therapy.

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Development and optimization of a pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants

Plant-virus-derived vectors are versatile tools with multiple applications in agricultural and medical biotechnology. In this study, we developed pepino mosaic virus (PepMV) (family Alphaflexiviridae; genus Potexvirus) into a vector for heterologous protein expression in plants. PepMV was initially cloned in a step-wise manner, fully sequenced and the full-length infectious clone was tested for infectivity in Nicotiana benthamiana. Initial infectious clones resulted in poor replication of PepMV and lack of systemic movement. Mutations in the viral sequence affected systemic infection. Two suspected mutations were altered to restore systemic infectivity. PepMV infection was apparent as early as 4 days post agroinfiltration (dpa) inoculation in N. benthamiana. A multiple cloning site was inserted into the PepMV genome for introduction and expression of foreign genes. Several modifications to the wild-type vector were made, such as a replacing the native subgenomic promoter (SGP) with a heterologous SGP, and introduction of translational enhancers and terminators, to improve heterologous expression of the foreign gene-of-interest. GFP was used as a reporter for monitoring virus infection and protein production. Strong GFP expression was observed as early as 4 dpa with a translational enhancer. The PepMV-based vector produces rapid expression of the foreign gene in comparison to two other potexvirus-based vectors. GFP production was monitored over time and optimal protein production was recorded between 5 and 7 dpa. GFP protein levels reached up to 4% and decreased to 0.5% total soluble protein at 7 and 14 dpa, respectively. Future studies will evaluate this virus-based vector for large-scale production of pharmaceutical compounds. KEY POINTS: • A pepino mosaic virus isolate was developed into a plant-based expression vector. • Expression levels of the heterologous protein were comparable or exceeded previously developed viral vectors. • Protein levels in plants were highest between 5 and 7 days and decreased gradually.

Sequence and characterization of shuttle vectors for molecular cloning in Porphyromonas, Bacteroides and related bacteria.

There is a lack of shuttle vectors to be needed for investigations into the genetics of Porphyromonas gingivalis and related species. To better understand the prevalence of candidates for such tools, we have examined multiple strains of black-pigmented anaerobes (clinical and laboratory isolates) for plasmids. As no plasmids were found in P. gingivalis strains, we have used the pYH420 plasmid, derived from P. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 from Escherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that it contained a gene with similarity to rep from plasmid pTS1 (isolated from Treponema denticola) as well as a homolog of mobA, a member of a gene family found on mobilizable genetic elements found in the genus Bacteroides. We constructed the pG106 and pG108 shuttle vectors using parts of the pUC19 and pYH420 vectors. This resulted in a vector with a multiple cloning site (MCS) in the lacZ gene enabling us to perform blue-white colony selection. The pG106 and pG108 shuttle vectors are electro-transformable into E. coli, P. gingivalis and B. thetaiotaomicron, where they are stable. We demonstrated that these vectors were suitable in these species for applications of molecular cloning including complementation and gene expression studies. Using the pG108 vector, we complement the hcpR mutant strain of P. gingivalis and rescued its -sensitive phenotype. We also performed a gene expression study using the P-glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.

Abstract B11: Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila

Abstract Colorectal cancer (CRC) is an extremely complex and genetically diverse disease with few available models that recapitulate the key features of heterogeneity. The Cancer Genome Atlas has identified 30 recurrent mutated genes as drivers of CRC. Individual tumors can carry any number of combinations of these mutations. A critical challenge in research has been the ability to model these genetically heterogenous tumors in animal systems. Highly sophisticated genetic tools in Drosophila make it an ideal model system to create and study different genome-based CRC tumors that well complement other animal models. Highly conserved signaling pathways and high similarity in gut homeostasis also make it a powerful and representative system to better understand CRC tumors and their progression. We have previously generated a panel of genetically complex Drosophila models that reflect the genomic landscape of sequenced colon tumors. The most complex model in this panel carried transgenic constructs to knock down Drosophila orthologs of tumor suppressors TP53, APC, SMAD4, and PTEN as well as to express oncogenic KRASG12V. However, creating more complex models using standard Drosophila crosses has been challenging. To address this, we have generated a multigenic vector that carries three different multiple cloning sites (MCSs) designed for cloning three separate transgenes. Two are designed for inducible protein expression to model oncogenic activation while the third is for a micro-RNA inspired synthetic multi-hairpin cluster to knock down multiple tumor suppressors. Here, we will report our efforts to further validate and expand this technology to build more complex cancer models. Using a series of test constructs, we show that transgenic flies carrying synthetic clusters of as many as 16 hairpins can be generated. Hairpins expressed in the context of synthetic clusters are as effective as single hairpins with no detectable positional effects. Finally, by introducing additional design elements such as intron-mediated hairpin cluster expression and bicistronic protein expression using a T2A self-cleaving peptide design, we have generated transgenes that express two proteins and up to 16 hairpins from a single transcript. We then used this updated platform to build a proof-of-concept colorectal cancer model that reflects IRS2 amplification, oncogenic KRASG12V, and loss of tumor suppressors TP53, APC, SMAD4, and SMAD2 from a single transcript designed for inducible expression. This new design frees up the remaining two cloning sites in our multigenic vector for additional genetic manipulations and pushes the limits of our technology to build a new generation of colorectal cancer models to study features of human tumors that have been traditionally challenging to capture in experimental models. Using this technology, we hope to better understand tumor development and find potential therapies that can specifically target these unique, personalized CRC genome-based models. Citation Format: Maria Quintero, Alexander Teague, Ross Cagan, Erdem Bangi. Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B11.

Biochemical and Molecular Characterization of Pichia pastoris Cells Expressing Multiple TMOF Genes (tmfA) for Mosquito Larval Control.

Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of Aedes aegypti, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in Chlorella dessicata and in Saccharomyces cerevisiae cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, Pichia pastoris, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. P. pastoris was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: gfp-tmfA and tmfA inserted between KpnI and XbaI in the multiple cloning site. The plasmid carries a strong AOXI promoter and P. pastoris KM71 and KM71H cells were transformed by homologous recombination. The synthesis of GFP-TMOF was followed using UV and clones were analyzed using southern and Northern blot analyses. Cloning tmfA into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of tmfA. A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a tmfA transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with gfp-tmfA identified a protein band that ran at the expected Mr of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 108 and 8.27 × 107 cells produce 229 and 114 μM of TMOF, respectively, and caused 100% larval mortality when fed to groups of 5 larvae in 25 mL water. These results indicate that the recombinant P. pastoris cells could be used in the future in the marsh to control mosquito populations.

Shikimate Kinase Expression; Towards Screening of Potential Antibacterials from Marine Sponges

Tuberculosis is one of the world’s leading cause of mortality from a single bacterial pathogen, with over 10 million reported cases each year. There is an alarming increase in the prevalence of drug‐resistant strains, thus the need for the discovery of novel anti‐tubercular agents. The shikimate pathway is a seven‐step metabolic route that produces aromatic amino acids and other cellular metabolites. It has no mammalian counterpart, making any of the enzymes in this pathway suitable targets for screening of potential anti‐tubercular agents. Mycobacterium tuberculosis shikimate kinase (MtSK) catalyzes the 5th step of this pathway, converting shikimate to shikimate‐3‐phosphate using ATP as a co‐substrate. The overall goal of this project is to express and characterize MtSK and screen for potential anti‐tubercular agents. Transformation of XL‐1 blue competent cells was performed using a pET 21b plasmid with aroK gene inserted at the multiple cloning site. Plasmids were cloned and purified and used to transform BL 21 DE3 competent cells for subsequent protein expression. Small scale expression showed the presence of a band of increasing prominence around 20 kDa. This suggests MtSK was successfully expressed. Expression analysis for larger scale supported data from small scale. Purification, characterization, and MtSK kinetic parameters would be determined prior to enzyme inhibition studies using inhibitors like avarone (below), a marine sponge sesquiterpene quinone and derivatives thereof.Support or Funding InformationEast Stroudsburg University of PennsylvaniaAvaroneFigure 1

Development of a Simple and Quick Method to Assess Base Editing in Human Cells

Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

Synthetic multi-antibiotic resistant plasmids in plant-associated bacteria from agricultural soils

ObjectivesUnlike higher organisms such as domestic animals and cultivated plants, which display a robust reproductive isolation and limited dispersal ability, microbes exhibit an extremely promiscuous gene flow and can rapidly disperse across the planet by multiple ways. Thus, microbial plasmids, including synthetic replicons, containing antibiotic resistance genes are a serious risk to public health. In this short communication, we explored the presence of synthetic elements in alfalfa symbionts (Ensifer meliloti strains) from agricultural soils. MethodsA total of 148 E. meliloti isolates from alfalfa plants growing under field conditions were collected from January 2015 to June 2019. Antimicrobial susceptibility testing was performed under laboratory conditions. We identified five kanamycin-resistant E. meliloti strains (named K1-K5). Whole genome sequencing analysis and conjugations were used to identify and study the plasmids of K strains. ResultsWe found that the genomes of K strains contain ampicillin, kanamycin and tetracycline resistance genes, the reporter gene lacZ from Escherichia coli and multiple cloning sites. These sequences were found within <58-kb plasmids related to the self-transmissible IncP plasmid RP4 from human pathogen Pseudomonas aeruginosa. Conjugation experiments confirmed the ability of K strains to transfer antibiotic resistance via conjugation to the Pseudomonas background. ConclusionIn addition to the traditional analysis of plant growth-promoting factors, the commercial deregulation of putative natural inoculants should also include genomic studies to ensure a reasonable balance between innovation and caution.

A doxycycline-inducible C17.2 neural stem cell-based combination of differentiation and suicide gene therapy for an in vitro tumorigenic C6 glioma model

Neural stem cell (NSC)-based gene therapies have been recently developed as effective strategies for treating brain tumors through inherent tumor tropism. However, this methodology has two considerable challenges: preventing NSCs from dying from the therapeutic agents encoded by their equipped genes before reaching tumor sites and the clearance of exogenous NSCs after therapeutic treatments. For these purposes, we established a novel doxycycline-inducible retroviral plasmid pTRE3G-TKGFP. A herpes simplex type 1 thymidine kinase (HSV1TK)-green fluorescent protein (GFP) fusion protein coding sequence was integrated into the multiple cloning site II (MCS II) of the pQcXIX vector, and the CMV IE promoter (PCMV IE) of pQcXIX was replaced with the doxycycline-inducible TRE3G promoter (PTRE3G). We then cloned the coding sequences of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and soluble interleukin 6 receptor (sIL-6R) into MCS I to combine HSV1TK (TK)/ganciclovir (GCV)-based suicide gene therapy against tumorigenic C6 glioma cells. TNF-α, IL-6 and sIL6R could efficiently induce tumorigenic C6 glioma cell differentiation, resulted in down-regulating the cell proliferation rate and the tumorigenicity of glioma cells and up-regulating the production of differentiation markers, such as connexin-43. Furthermore, gap junctions could enhance the bystander effect in suicide gene therapy. Consequently, we found that the retroviral plasmid transfected NSCs exerted stronger remedial effects on tumorigenic C6 glioma cells through the combination of differentiation and suicide gene therapy than by suicide gene therapy alone. This study is also the first case applying NSCs to conduct the combination of differentiation and TK/GCV-based suicide gene therapy on glioma cells.

A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes.

Bacteria play an important role in the catabolism of environmental xenobiotics. The study of transcriptional regulation has greatly enhanced our understanding of the molecular mechanisms associated with these processes. However, genes encoding transcription factors (TFs) for xenobiotic catabolism are usually not located in the immediate vicinity of the catabolic genes that they regulate; therefore, functional identification of these TFs is difficult. Significantly modified from a metagenome screening method substrate-induced gene expression (SIGEX), here we propose a synthetic pSRGFP-18 plasmid-based tool as a TF reporter system. In short, two multiple cloning sites (MCS) were designed; one in front of an egfp reporter gene was constructed for promoter insertion, and the other MCS was used for shotgun cloning of genomic DNA. Based on the regulatory relationship between TFs and the promoter of their associated catabolic genes, this approach allowed us to screen for TF genes using a genome shotgun approach. This system performed well when testing the known operons. Following statistical analysis of known catabolic operons in Escherichia coli and Bacillus subtilis, the suggested region of the target promoter for this system was from - 250 to + 150. Furthermore, to broaden the applicability of this plasmid, a series of pSRGFP-18 and pBBR1-based pSRGFP-X plasmids were constructed, which had broad host ranges and contained different antibiotic markers. This study outlines a powerful tool to enable functional identification of TFs for bacterial xenobiotic catabolism.

Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2 Gene on SHI-1 Cell Growth

To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1). The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group. The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05). The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.

A novel expression vector for Corynebacterium glutamicum with an auxotrophy complementation system

Corynebacterium glutamicum is an important industrial strain used for the production of amino acids and vitamins. Most tools developed for overexpression of genes in C. glutamicum are based on the inducible promoter regulated by the lacIq gene or contain an antibiotic resistance gene as a selection marker. These vectors are essential for rapid identification of recombinant strains and detailed study of gene functions, but, as a considerable disadvantage, these vectors are not suitable for large-scale industrial production due to the need for the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) and antibiotics. In this study, the novel Escherichia coli-C. glutamicum shuttle expression vector pLY-4, derived from the expression vector pXMJ19, was constructed. The constitutive vector pLY-4 contains a large multiple cloning site, the strong promoter tacM and two selective markers: the original chloramphenicol resistance gene cat is used for molecular cloning operations, and the auxotrophy complementation marker alr, which can be stably replicated in the auxotrophic host strain without antibiotic selection pressure, is used for industrial fermentation. Heterologous expression of the gapC gene using the vector pLY-4 in C. glutamicum for L-methionine production indicated the potential application of pLY-4 in the development of C. glutamicum strain engineering and industrial fermentation.

A New Escherichia coli Entry Vector Series (pIIS18) for Seamless Gene Cloning Using Type IIS Restriction Enzymes.

A series of new Escherichia coli entry vectors (pIIS18-SapI, pIIS18-BsmBI, pIIS18-BsaI, pIIS18-BfuAI-1, and pIIS18-BfuAI-2) was constructed based on a modified pUC18 backbone, which carried newly designed multiple cloning sites, consisting of two facing type IIS enzyme cleavage sites and one blunt-end enzyme cleavage site. These vectors are useful for seamless gene cloning.

Utilization of a Multiple Cloning Site as a Versatile Platform for DNA Triblock Copolymer Synthesis.

DNA-containing block copolymers have utility in a wide range of biomedical applications. However, synthesis of these hybrid materials, especially ones with complex chain structures, remains to be a major challenge. Here, we report the use of a combination of restriction enzyme sites and ligation enzymes to synthesize DNA triblock copolymers. In contrast to triblock structures held together by DNA hybridization, the newly synthesized DNA triblocks have all blocks connected by covalent bonds. The improved stability of the triblocks against environmental factors such as urea denaturing is confirmed. Furthermore, we incorporate a multiple cloning site (MCS) into the DNA block copolymers and show that the restriction sites can be cut by their corresponding restriction enzymes, generating diblocks with different sticky ends. By utilizing these sticky ends of specific sequences, the cut diblocks are further ligated to create a variety of triblock copolymers with different DNA center blocks and synthetic polymer end blocks. This study presents a versatile platform based on MCS for the synthesis and regeneration of a range of DNA-containing block copolymers.

Expression and presentation of immune-related membrane proteins of fish by a cell surface display platform using insect cells

Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with “quality control” system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu’s CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen’s egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals.

Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration.

Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these "ICE" vectors, for "Integrating after CEN Excision." The ICE series was created by introducing a "rare-cutter" NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.

New \u03b1- and SIN \u03b3-retrovectors for safe transduction and specific transgene expression in pancreatic \u03b2 cell lines

BackgroundViral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing β cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into β cells.ResultsWe here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian β cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in β over non-β cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human β cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human β cell lines.ConclusionOur novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian β cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of β cell function and monitor changes in their differentiation status.