Dissertations / Theses on the topic 'Cloning vector'

Naegleria fowleri is a free-living amoeba that causes primary amoebic meningoencephalitis (PAM). In the United States, there are between 0-8 cases of PAM per year, with approximately 98% of cases resulting in death. High case fatality and limited treatment options highlight the need for better understanding of this organism in terms of its biology and pathogenicity. Transfection is a useful tool that allows for the study of gene function, but at present no transfection systems have been established for N. fowleri. This study attempts to establish a transfection system for N. fowleri using the piggyBac vector, with the hope of eventually using the piggyBac transposon system to identify novel genes related to pathogenicity in N. fowleri. To accomplish this, 5’ and 3’ regulatory regions for genes in the N. fowleri genome were amplified and inserted into a piggyBac vector with a GFP reporter gene via molecular cloning, and vectors introduced to the amoeba via electroporation. Although no GFP was visualized after transfection, there are several routes for optimization of the transfection system that could be explored. Development of a transfection system could allow for the study of pathogenicity in vivo, by either utilizing the transposon system of piggyBac or the expression of reporter genes for visualization of amoeba during the course of infection. Further elucidating N. fowleri pathogenicity factors could reveal new drug targets, give new information about the organism’s biology, and help better define an effective treatment regimen to combat PAM.

Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus.<br>Department of Biology

Background: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods: First, we modified the Tet-pLKO-Puro vector to make it easy("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.

A hybrid vector, pBVl, has been constructed which is capable of regulating expression of cloned genes in both Escherichia coli and in cyanobacterium Synechocystis 6803. Vector pBVl contains the powerful rightward promoter of bacteriophage lambda to ensure that cloned genes are transcibed at a high level. In addition, pBV] also contains a gene, cI857, encoding the lambda temperature sensitive repressor protein.The CAT gene coding for chloramphenicol aceyltransferase (CmActase) was cloned into pBV1 downstream of the lambda regulatory features to make plasmid pTC1. The expression of the CAT gene was quantified spectrophotometrically following addition of chloramphenicol and a shift in the growth temperature of the cell culture. The specific activity of CmActase increased from 540 to 5400 units within 30 minutes. Thus, using the newly constructed hybrid vector, pBVl, temperature regulation of gene expression in E. coli was observed.<br>Department of Biology

Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.

Published information on the structure and regulation of H. pylori promoters is limited. The work presented in this thesis describes the cloning and characterisation of promoter regions from a clinical isolate of H. pylori, and the development of an alternative, non-radioactive method for verifying the location of transcriptional start sites of bacterial promoters. H. pylori 4187E promoters were randomly cloned into the promoter-trap vector pKK232-8 in Escherichia coli DH5α using two sets of restriction enzymes. Vector pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene. Seventy-four promoter-containing clones were isolated from selective media based on their resistance to chloramphenicol. The strength of each promoter was analysed qualitatively, using chloramphenicol minimum inhibitory concentrations, and quantitatively, using CAT assays following exposure of the clones to pH 4 and pH 7. Selected promoter fragments were subcloned into the GFP reporter vector pFPV25, containing a promoterless gfp gene. The subclones were exposed to buffered LB broth at pH 4, 5, 6, 7 and 8, for varying lengths of time, to study acid-induced regulation of gene expression. Subclones were examined qualitatively, using visual examination of GFP fluorescence and fluorescence microscopy, and quantitatively, using flow cytometry following acid shock. DNA sequences were determined for 61 of the 74 H. pylori promoters, and sequence alignments with the published H. pylori strains (26695 and J99) were performed. The transcriptional start site of 27 H. pylori promoter fragments was experimentally mapped using a fluorescence-based primer extension protocol developed by our group. Potential -35 and -10 sequences were identified for each promoter, and a new consensus sequence for H. pylori promoters was proposed based upon these results. This study has considerably expanded knowledge of H. pylori promoter sequences and transcriptional start sites based on those which also function in E. coli. It has also revealed several H. pylori promoters which appear to respond to acid stress

A hybrid vector, pANIC1, was to be constructed which was capable of regulating expression of cloned genes in both Escherichia coli and Anacystis nidulans R2 by controlling the iron content of the growth medium. Plasmid pANIC1 would have origins of replication for E. coli and A. nidulans R2, and a marker gene conferring ampicillin resistance. It would also contain the promoter for the irpA gene which is active only in low iron growth conditions.The first two stages of the construction were successfully completed, but unfortunately the final construction proved to be unstable. Recent information has shown that operator sequences upstream from the irpA gene's promoter result in an unstable message. This may be interfering with the normal functioning of the host cell, resulting in an unstable construction. In future experiments it may be neccessary to alter the growth conditions or remove the upstream sequences in order to stablize the construction.<br>Department of Biology

Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts. Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.

Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant.<br>Ph. D.

Recherche d'outils génétiques utilisables chez la bactérie pseudomonas fluorescens MFO et mise au point des conditions de leur utilisation aux différentes températures de croissance de la souche. Mise au point d'un vecteur de clonage, de conditions d'intégration de gènes étrangers dans le chromosome bactérien, d'une méthode de cartographie par transfert chromosomique et de transposition par génétique réverse

The fusion yeast, <I>Schizosaccharomyces pombe, </I>has several potential advantages as a host for a cloning system for large DNA molecules when compared to the budding yeast, <I>Saccharomyces cerevisiae. </I>The possibility of constructing a large DNA cloning system in <I>S. pombe </I>has been investigated. <I>S. pombe </I>has 3 large chromosomes (greater than 3.5 Mb) and may therefore have the ability to carry megabase sized artificial chromosomes. In addition artificial chromosomes of less than 3.5 Mb will migrate below the host chromosomes on pulsed field gels and thus will be easier to separate away from the host chromosomes. As in higher eukaryotes, the <I>S. pombe </I>centromeres have arrays of repetitive elements, therefore such sequences from heterologous sources which rearrange in <I>S. cerevisiae </I>may not do so in <I>S. pombe. </I>It has been shown that small acentric linearised plasmids with cloned <I>S. pombe </I>telomeres at each end replicate intact in <I>S. pombe. </I>These plasmids were used to prepare vector arms each having a selectable marker, <I>S. pombe </I>telomere and <I>S. pombe </I>replication origin. The <I>S.</I><I> pombe</I> artificial chromosome (SPARC) vectors do not contain any <I>S. pombe </I>centromere sequences as they are not necessary for the maintenance of the SPARCs under selection. To test the potential of the SPARC vectors for cloning human sequences in <I>S. pombe, </I>well characterised human fragments derived from cosmids were cloned in <I>S. pombe </I>using these SPARC vectors.

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Sanidade Animal<br>A modulação de imunidade específica por conjugação de antigénios com ligandos de receptores de reconhecimento de padrão (PRR) constitui uma estratégia emergente para o desenvolvimento de vacinas subunitárias. Neste trabalho, desenvolve-se um novo sistema de clonagem em Escherichia coli para expressão de antigénios em fusão com a lipoproteína OprI, um ligando TLR da membrana externa de Pseudomonas aeruginosa. O sistema permite um controlo apertado da expressão proteica e a purificação por cromatografia de afinidade com iões metálicos, ultrapassando as principais limitações de versões anteriores. Confirmou-se o processamento, translocação e triacilação da lipoproteína e desenvolveram-se protocolos para a produção de outras formulações recombinantes derivadas da parede bacteriana (fragmentos e vesículas de membrana externa) com potencial distinto para activação PRR. Como modelo, clonaram-se as sequências dos antigénios A104R do vírus da peste suína africana (VPSA), ovalbumina e EGFP. Demonstrou-se a capacidade adjuvante das três formulações, avaliando a resposta humoral e a indução de linfócitos T CD8+ in vivo e o perfil de citocinas e quimiocinas induzidas em células dendríticas estimuladas in vitro. Os resultados observados validam o sistema para a obtenção de formulações imunogénicas com aplicação no desenvolvimento de vacinas subunitárias experimentais e em estudos de modulação de resposta adaptativa.<br>ABSTRACT - A new cloning system based on the OprI lipoprotein for the production of bacterial cell wall-derived immunogenic formulations - The modulation of specific immunity through the conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the development of subunit vaccines. Here, a new Escherichia coli cloning system for the expression of antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane, is described. The system offers tight regulation of expression and allows for purification by metal affinity chromatography, circumventing the major drawbacks of former versions. Lipoprotein processing, translocation and triacylation were confirmed and protocols for the productions of other recombinant bacterial cell wall-derived formulations (outer membrane fragments and vesicles) with distinct potential for PRR activation were developed. As models, the sequences coding for the antigens A104R from African swine fever virus (ASFV), ovalbumin and EGFP were cloned. The adjuvant capacity of the three formulations was demonstrated evaluating the induction of humoral and CD8+ T cells responses in vivo and the cytokine and chemokine profile induced in dendritic cells stimulated in vitro. The results observed validate the system for the production of immunogenic formulations suitable for the development of experimental subunit vaccines and for studies on the modulation of adaptive immunity.<br>FCT-Fundação para a Ciência e Tecnologia. CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal da Faculdade de Medicina Veterinária-UTL.

The 33 kilodalton (kD) manganese stabilizing protein (MSP) is intimately associated with the photolysis of water to molecular oxygen. The two main purposes of this study were: 1) to analyze previously constructed MSP mutants and 2) to subclone, express, and purify the wild-type MSP in Escherichia coli in order to investigate the relationship between structure and function of this protein.Growth rates were compared between bacterial cells harboring only the vector, the vector plus the wild-type MSP gene, and the vector plus a mutant MSP gene. No significant differences were seen. This result implies that the expression of the wild-type MSP or mutant MSP is not toxic to the cells. Plasmid DNA isolation and restriction analyses of several of the mutant clones also confirmed the presence of the correct size inserts in the vector. However, upon sequencing several mutant clones, it appeared that losses and/or rearrangements of sequences was occurring. Thus, it was concluded that MSP was not being stably maintained in E. coli.Expression of the wild-type gene was achieved in E. coli by IPTG induction of the gene in pUC120 cloned under the control of the lac promoter. The expressed protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and confirmed by western blotting. Purification of the wild-type protein was obtained by membrane fractionation over a DEAE ion exchange column and the expression product was detected by western blotting. However, the expression product was lost in the concentration procedure and therefore is not available for reconstitution experiments.The wild-type MSP gene was also subcloned in a hybrid shuttle vector pTNTV, previously constructed in our laboratory (1). This construct was used to permit constitutive highlevel expression of the cloned gene and may prove to be an alternative vector to better express the MSP and mutant MSP in future investigations.These results demonstrate that it is possible to express the wild-type MSP gene from cyanobacteria in E. coli, but the problems of instability and recombination of the mutant genes in the vector have to be addressed before proper expression of these genes can be obtained.<br>Department of Biology

A cotton PATE cDNA clone has a 1.7-kb insert with an coding region for 410 amino acids, lacking codons for the three N-terminal amino acids. The predicted amino acid sequence of the PATE preprotein has a characteristic stromal-targeting domain and a 63% identity to the Arabidopsis FatB1 thioesterase sequence. A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-ACP thioesterase (FatB1) gene. The gene spans 3.6 kb with six exons and five introns. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein is identified as a FatB thioesterase from its deduced amino acid sequence similarity to those of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential promoter/enhancer elements including basic helix-loop-helix elements (E box). Alkaline blot hybridization of cotton genomic DNA suggests the presence at least two FatB1 thioesterase genes in cotton. Four plasmid constructs for both constitutive and seed-specific anti-sense RNA suppression and gene-transgene co- suppression of PATE gene expression were successfully generated. Two overlapping cotton genomic clones were found to encompass a Δ-12 fatty acid desaturase (FAD2-3) gene. The continuous FAD2-3 coding region is 1,155 bp and would encode a protein of 384 amino acids. The FAD2-3 gene has one large intron of 2,967 bp entirely within its 5'-untranslated region. Several potential promoter/enhancer elements, including several light responsive motifs occur in the 5'-flanking region. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro.<br>Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

碩士<br>國立清華大學<br>分子醫學研究所<br>92<br>One new strain of Lactobacillus, identified as Lactobacillus rhamnosus TCELL-1, was isolated from the healthy adult rectum biopsies in our laboratory. A new food-grade shuttle cloning vector for Lb. rhamnosus TCELL-1 and E. coli was constructed using the nisin immunity gene nisI as a selection marker. The food-grade shuttle cloning vector, pNI, was constructed using the pAMβ1 replicon, the pUC origin, the nisI gene, the promoter Lac for nisI expression, and the nucT reporter gene. Electroporation into Lb. rhamnosus TCELL-1 was selected with the nucT reporter gene. Plasmid pNI was confirmed in Lb. rhamnosus TCELL-1 with southern hybridization. Lb. rhamnosus TCELL-1 carrying pNI was shown to be able to grow in medium containing a maximum of 60 IU nisin/ml. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in Lb. rhamnosus TCELL-1 in order to construct improved starter bacteria for food applications.

碩士<br>國立清華大學<br>生物技術研究所<br>90<br>Lactic acid bacteria (LAB) are Gram-positive bacteria and generally regarded as safe (GRAS) organisms. LAB could be used for heterologous protein secretion and fused antigen to form fusion protein. They are good potential candidates as antigen delivery vehicles. We developed an efficient secretion system in the Lactococcus lactis model. Staphylococcal nuclease (Nuc protein) is a small, stable, and biochemically well-characterized enzyme secreted by Gram-positive bacteria. It was used as the reporter protein. Plasmid pNuc10 encoding LEISSTCDA-NucT is a L. lactis cloning vector. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). A 9-residue synthetic propeptide, LEISSTCDA , was fused immediately after the signal peptide cleavage site. Secretion efficiency was increased to ~90% by LEISSTCDA insertion without altering the signal peptide cleavage site. Because the cloning vector, pNuc10, could not replicate in E.coli. We fused pBluescriptⅡSK(+) and pNuc10 to form the shutter vector pBN. We transformed DNA to E.coli by heat shock, and it would replicate in supercoil form for ColE1 origin of E.coli. Then the expression vector is transferred to the L. lactis MG1363 strain by electroporation. In this study, the gp25 glycoprotein of EB virus was fused with histidine tag as a heterologous protein, and was cloned between NucT and LEISSTCDA to form fusion protein. We hope that the L.lactis MG1363 transformed with this expression vector can be used as a live vaccine system.

碩士<br>國立清華大學<br>生物技術研究所<br>91<br>Our Laboratory has isolated a new strain of Lactobacillus rhamnosus, named T CELL-1, from intestines of a healthy adult in Taiwan. Unfortunately, we were unable to detect any endogenous plasmid in it. For DNA manipulation, it is necessary to construct a plasmid which has a origin that allow it to replicate in Lb. rhamnosus T CELL-1.Therefore, we screened all other Lactobacilli in our Laboratory collection for native plasmid and tested whether they can replicate in Lb. rhamnosus T CELL-1. As a result, we found a plasmid, pLP1, being suitable as a cloning vector, from one of the 13 strains. The plasmid was amplified pLP1 by inverse PCR and constructed into a shuttle vector, pBLP, which could replicate in both E. coli and Lactobacilli. An antibiotic-resistance gene, ermC, was inserted as a selection marker and the resulting plasmid was named pBLPE. The recombinant plasmid was subsequently transformed into Lb. rhamnosus T CELL-1 by eletroporation. The best condition for eletroporation mediated transformation of this Lactobacillus was also determined. Plasmid pBLPE is somewhat unstable in Lb. rhamnosus T CELL-1 and no complete plasmid was detected. To solve the problem of plasmid instability, we deleted the region derived from E. coli and self- ligated remaining portion to produce a new recombinant DNA, pLPE. The latter, pLPE, was more stable than the former, pBLPE. The experiment revealed that it had a problem of structure after eletrotransformation and the sequence would be changed, with Southern hybridization and several primers of PCR. Nevertheless, some minor sequence alterations of the plasmid also detected after it was introduced into Lb. rhamnosus TCELL-1.The details of this sequence change remain to be identified.

碩士<br>國立陽明大學<br>神經科學研究所<br>90<br>Kv4.2, a voltage-gated K+ channel gene, is most abundantly expressed in the hippocampus. In order to examine whether Kv4.2 is involved in learning and memory, we plan to establish a line of conditional knockout mouse in which Kv4.2 is deficient in the hippocampal neurons. The first step is to isolate the genomic clone of Kv4.2 gene. In this study, using the 5’non-coding region of rat Kv4.2 cDNA as probe (a 452 bp PCR fragment), we screened a S129 mouse genomic phage library by Southern hybridazation. One positive phage clone was isolated and the size of insert is 16,197 bp. After sequence analysis, the phage insert contained the 5’-flanking sequence (5.1 kb), exon 1 (2 kb) and part of intron 1 (8.9 kb) of mouse Kv4.2 gene. The exon 1 contains 60% of the Kv4.2 total protein sequence. The second step, to construct the targeting vector for conditional knockout, we used pBS246 and pKO910 vectors as the backbones to subclone various DNA fragments. Hypoxanthine phosphoribosyl transferase gene (hprt) and diphtheria toxin gene (DT) were used as the recessive and the dominant selection markers, respectively. The 5’-long arm (5.6 kb, containing the 5’-flanking sequence, exon 1 and part of intron 1 inserted between two loxP sites) will be the fragment for deletion. Same as the orientation of transcription, the short arm (2.5 kb, containing the more distal 5’-flanking sequence), and the 3’-long arm (2.7 kb, containing part of intron 1, connected after the DT gene) were respectively subcloned into the DT-pKO910 vector for homologous recombination.

碩士<br>中國文化大學<br>生物科技研究所<br>80<br>F9細胞株是一種未分化之老鼠腫瘤細胞，此種細胞可被一些物質誘導分化如DMSO，荷 爾蒙和維生素A 酸。F9細胞株被誘導分化後有些基因會大量表現，有些基因表現量則 會大幅下降，但這些基因在F9細胞株和分化過程所扮演的角色不詳。Krishnamoothi et al.雖分離了F9細胞中一段具有加強子功能的DNA ，但在整個分子層次上，究竟什 麼因子控制F9維持未分化狀態前目前也不明瞭。所以我們想利用Promoter trap 的方 法來分離一些F9細胞中之特殊基因。 Promoter trap 是利用一些不帶有起動子的報導基因質體，送至細胞中來鑑定寄主細 胞中的起動子。我們構築一個具有功能的反轉錄病毒載體，將它送入F9細胞中來鑑定 其染色體中的起動子。我們共得到了九種不同的flanking DNA，而根據定序的結果， 發現言些flanking DNA中的GF-1和GF-2定序序列各別與老鼠中的U6 snRNA及LINE-1基 因有極高的相似性；另外在GF-7的定序中則有段23次的"GT"重覆序列。這些flanking DNA究竟在F9細胞中扮演什麼角色，則要更進一步的分析。

碩士<br>國立清華大學<br>輻射生物研究所<br>81<br>Cloning vector construction and a plasmid-free recipient isolation must be accomplished for developing the gene cloning system in Deinococcus. Two radiation-sensitive mutants, D. radiodurasns S101 and D. radiodurans PF3-3, were isolated from MNNG-mutagenized D. radiodurans strain IR. Both also cured the 28 kb pST7 which is the cryptic plasmid of strain IR and the spontaneous reversion frequencies of them to radiation resistance were 1--5 x 10^-8. This indicated that the plasmid of strain IR was not necessary for radiation resistance. The native transform -ability of strain S101 was 1000-fold higher than IR's, and strain PF3-3 was similar to strain IR's. An Escherichia coli plasmid pBR328, that confers Cmr ( chloramphenicol ) resistance, was ligated with pST7 to yield the 33 kb hybrid plasmid, pIR7. pIR7 transformed various E. coli strains at low frequencies and existed unstably in E. coli strains. When pIR7 transformed strain IR, the pST7 disappeared and only a 6.6 kb recombinant plasmid, designated pCMR, could be detected in these Cmr transformants. We assumed that pCMR was generated by rearrangement between pIR7 and pST7. pCMR was able to transform D. radiodurans plasmid-free strains, strain S101 and strain PF3-3, to Cmr at high frequencies and remained unchanged through these transfers. Various deleted plasmids were isolated from E. coli which transformed with pCMR. Only pCM5 (sized 5.4 kb) of deleted plasmids demonstrated to be Cmr, but it could not transform D. radiodurans strains any more. Conclusions of above results, (i) pCMR should carry the replication element of pST7 besides the selection marker, cat gene, and it is potential to be a cloning vector for D. radiodurans; (ii) there were some barriers existed in plasmid transformation between D. radiodurans and E. coli; (iii) the plasmid-free strain,strain PF3-3, might be an appropriate recipient for plasmid transformation in D. radiodurans; (iv) the plasmid of D. radiodurans IR was not necessary

碩士<br>國立清華大學<br>生命科學研究所<br>82<br>HIV IN 為逆轉錄病毒早期生活史中重要之蛋白質 , 它負責了將病毒的核 酸序列插入寄主的染色體中, 形成潛伏病毒;文獻指出 HIV IN 在許多 in vitro 的實驗中 , 已被証實可以單獨執行核酸序列銜接的工作。 而 本篇實驗中所欲探討的是 : IN 若可以在 in vivo 狀態下 ,單獨存在、 且執行功能 , 其首先必須進入細胞核中 。 就多數的核蛋言 : 已有許多 報告分別以採取了刪除/定位突變/ 遺傳工程及化學合成融合蛋白等策略 來模擬核傳送訊號序列(NLS)的活性 , 指出核蛋白本含有的NLS 可使細胞 座落於核中;諸如此例可見於 SV40 large T antigen﹑p53﹑Polyoma Virus VP1等.. 這種具有NLS的活性序列經分析的結果得知: 大多數由數 個鹼性胺基酸所組成的短鏈, 同時我們在 IN 這個蛋白序列中發現疑似這 種NLS 活性的序列於是我們希望進一步藉由突變的方式來鑑別它的存在。 首先我們建構了一個以Bovine Papilloma Virus Vector 為表現的載體系 統, 將 HIV-I IN 載入上述質體中,此表現系統屬於 Episomal mammalian expression system,由來自金屬硫蛋白(MT)基因之啟動子 (promoter)、Moloney murine Sarcoma Virus 之末端重覆序列 (LTR) 為 促進子 (enhancer) 、 以及 SV40 之 mRNA splicing junction 和 polyadenylation signal 來幫助插入基因有效表現於真核細胞中。同時 此載體也帶有複製所必須之 ori 及來自 pBR322 之 β lactamase之基 因, 使其成為一穿梭載體 。 由於所插入基因與質體大小相距太大(0.8 kb:12.5kb)致使直接的載入的工作不易進行;故先行將病毒基因(BPV)的部 分去除,建構一個只在大腸桿菌中表現的小載體 (4.5kb),將IN基因載入, 成為4571IN, 再將此成功導入之載體接回原來刪除之病毒基因。 此外,也 同樣的針對IN上疑似NLS的序列( 261-267 amino acids) ,做定位突變, 預期在將來送入細胞(3T3/C127)後,以免疫沉澱的方式,追蹤其表現所座落 的區位是否受到改變,來做為鑑別NLS的根據。

Thesis (M.S.)--University of Wisconsin--Madison, 1990.<br>Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 36-42).

A shuttle vector was constructed which was stably maintained in Escherichia coli, Bacillus subtilis and Staphylococcus aureus. It was made by ligating E. coli positive selection plasmid pEcoR251 and S. aureus resistance plasmid pC194 via their respective BamHI and XhoII sites. Designated pNDW1, it was shown to be effective in genomic library construction. The number of restriction sites in the EcoRI (“suicide”) gene was increased by successive addition of XbaI and XhoI using site-directed mutagenesis. SpeI, NheI and AvrII generate DNA fragments with compatible cohesive ends to XbaI while SalI digestion gives rise to ends compatible with XhoI. Therefore the number of different genomic libraries which can be made using this system is augmented by six. A principal impediment to full exploitation of these shuttle vectors is apparently the severe restriction by B. subtilis and S. aureus of DNA coming from E. coli.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1987.<br>Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 88-96).

博士<br>國立中興大學<br>獸醫學系<br>87<br>Most of cloning vectors used in Lactobacillus reuteri are adapted from systems already available in other microorganism and generally bear disadvantages of low efficiency and instability. Thus, the aim of this study was to search for some parts (i.e., antibiotic selection markers and replication regions) from indigenous plasmids of this microbe with good properties potentially used in constructing efficient cloning vectors for this bacteria. The final goal of these constructions is to provide tools for the molecular studies, the strain improvement, and the development as vaccine carrier of this bacteria species. This work began with the construction of physical maps from one chloramphenicol resistance (Cmr) plasmid (i. e., pTC82) and four erythromycin resistance (Emr) plasmids (i. e., pTE31, pTE32, pTE15, and pTE80) followed with sequencing determination and molecular analysis of their antibiotic resistance determinants. A total of 1746-bp, which include the Cmr determinant and its flanking region from the plasmid of pTC82, was sequenced. Further determination of the nucleotide sequence of the Cmr gene (cat-TC) on pTC82 revealed an open reading frame (ORF) for a 238-amino-acid Cm acetyltransferase (Cat) monomer. This structural cat-TC gene, 714-bp in length, was highly related (ca. 95% identity) to the cat gene from Staphylococcus aureus plasmid pC194. Immediately upstream to the Cat coding region, the likely genetic expression regulators including promoter, ribosome binding sites (RBS), and leader sequence were observed and were subsequently identified as a functional and inducible type of expression in this microbe. To determine the activity of this putative cat-TC gene, a maxicell analysis was conducted and was able to identify a synthesized protein with molecular weight in reasonable agreement with the prediction from the DNA sequence. To the four Emr determinants, totals of 1173-bp (pTE31), 1077-bp (pTE32), 1979-bp (pTE15), and 1076-bp (pTE80) were respectively sequenced. Determination of the nucleotide sequences of the genetic determinants encoding resistance to Em on the four plasmids revealed a very similar ORF for a 248 (or 250)-amino-acid rRNA methylase (Erm) existing in each of these plasmids. These putative structural erm genes, respectively called erm31, erm32, erm80 and erm15, were highly related (ca. 93-99 nucleotide identity) to the erm genes of E. coli pIP1527, Streptococcus faecalis Tn917, Streptococcus agalactiae pIP501 and Lactobacillus fermentum pLEM3 and were thus categorized under the ermB class. The activities of these putative erm genes were also proved to be functional by a maxicell analysis in which synthesized proteins with a molecular weight of 29.4 kDa, being in agreement with the prediction from the nucleotide sequences, were observed. The nucleotide sequences of cat and erm genes revealed that in these works are the first report in L. reuteri. For facilitating the cloning of replication region (RR) from the five plasmids, a RR-probe vector, namely pUE80 (+/-) was constructed by the ligation of the erm80 gene of pTE80 into the SspI site of pUC18 and pUC19 respectively. Five DNA fragments each containing a RR from the five plasmids, being in the length of 2.2-kb, 3.0-kb, 3.1-kb, 3.0-kb and 3.3-kb respectively, were successfully cloned into the pUE80 (-) to generate the recombinant plasmids of pTE31-RO, pTE32-RO, pTE15-RO, pTE80-RO, and pTC82-RO respectively. Further sequence analysis of these RRs revealed that the RR from pTE31, with the presence of elements of DSO (double strand origin), rep31 (replication initiation gene), and SSO (single strand origin) typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR) of pC194 family, was highly related (ca. 99% nucleotide identity) to the RR of pLEM3 from L. fermentum. Two more ORFs potentially encoding products, respectively, of 210- and 234-amino acid in length were also identifiable in the sequence of pTE31. Their functions remain to be determined. As to the nucleotide sequence of RRs from pTE32, pTE80, and pTC82, a highly sequence similarity was found among one another. Elements (i. e., DSO, rep, and SSO) with conservative characteristics of RCR plasmids of pC194 family were also detectable in these sequences. For examples, despite the low sequence similarities existing between these three RRs and other known RRs with respect to the flaking regions of the nic site of DSO, a conservative nucleotide sequence (CTTGATA) typical of the nic site of pC194 family was actually detectable in all three RRs. The putative rep genes from three RRs were predicted to encode proteins of a molecular weight of 86.7 kDa and 749 amino acids in length. Similar to the condition in nic site, despite little homologies (less than 23%) in amino sequence were found between these three Reps and other known Reps, a region containing three motifs typically found in Reps of pC194 family was also exactly identified in these three RRs. Analysis by employing the GCG software further indicated the deduced Reps as a novel protein belonging to the pC194 family. The SSOs from three RRs showed a strong homology of 66% to the typical sequence of SSOT type from pBAA1 of Bacillus subtilis. These are the first reported sequences of SSOT type in L. reuteri. Besides, three more ORFs, each with potential of encoding proteins of 419, 221, and 72 amino acids in length respectively, were also detected in the sequence of pTC82. To the deduced 419-amino acid product, the 214 amino acids located at the N-terminus was shown to share a homology of 40% in amino acid sequence with the mob gene of pNZ4000 and pCI528 from Lactococcus lactis. Functions of the other two deduced products are still cryptic. Nucleotide sequence of the RR from pTE15 revealed the presence of eight inverted repeats (IR), three AT-rich direct repeats (DR), and two ORFs (namely RepA and RepB). The amino acid sequences of the deduced RepA and RepB from pTE15 showed homologies of 57% and 32% respectively to those of RepB and RepC from Enterococcus faecalis plasmid pAD1. Through a series of analysis including the restriction-deletion study, the copy number detection, and the segregational stability study, functions of the deduced RepA and RepB from pTE15, although dispensable for the plasmid replication, were determined as regulators for plasmid copy number and segregational stability. Judging from the organization mentioned above as well as the absence of Rep protein essential for plasmid replication in the nucleotide sequence, it is tempted to categorize the RR from pTE15 under the θ(theta) type of replication resembling that of pLS20 from Bacillus subtilis and ColE1-type plasmids. However, addition of Cm into the growing culture of L. reuteri, which contained the plasmid pTE15 failed to demonstrate the significant amplification of pTE15. It may thus suggest that the replication of pTE15 is more likely to be the same as that of pLS20. By employing the technique of pulse-field gel electrophoresis (PFGE), a molecular size of 1.95-Mbp for the chromosome of L. reuteri DSM 20016 was obtained. With the availability of this chromosome size, the copy numbers for the recombinant plasmids of pTE31-RO, pTE82-RO, and pTE15-RO were able to be determined as 21, 13, and 2 respectively. As to the host spectrum analysis, plasmids pTE15-RO, pTE32-RO, pTE80-RO, and pTC82 were capable of replicating in L. reuteri only, whereas plasmid pTE31 was found to have a wider host spectrum including L. reuteri and L. fermentum. For the last item of plasmid characteristics analysis, namely the segregational stability, all the recombinant plasmids tested in this study had a stability index of 100% after 216 generations except plasmid pTE15-RO which had an stability index of only 4% after 144 generations. By employing materials including the erm80 gene, four RRs (from pTE15, pTE31, pTE32, and pGK12), and parts (ColE1, lacZ, and multiple cloning site) from pBluescript II SK (-), which were analyzed in this study thus far as bearing good qualities for the construction of vectors, four E. coli-L.reuteri shuttle vectors, named as pSTE31 (4.9-kb), pSTE32 (5.7-kb), pSTE15 (4.0-kb) and pSGK12-80 (4.2-kb) respectively, were successfully constructed. Further evaluation of these vectors revealed that (1) at least 13 unique restriction sites were available for each of the vectors, (2) a high stability index of 100% after 216 generations was observed for the vectors of pSTE31 and pSTE32, while low stability indexes of 4% and 3% after 144 and 180 generations were respectively obtained for vectors of pSTE15 and pSGK12-80, (3) an aprA gene from Bacillus subtilis and a cat gene were cloned into pSTE31 and pSGK12-80 respectively and were successfully expressed and secreted from the recipient host of L. reuteri. Furthermore, a promoter-screening vector (pSTE31-cat, 5.6-kb) and a transcriptional terminator-screening vector (pSTE31-catT, 5.9-kb) had also been constructed in this study. Hopefully, more materials such as promoters and terminators from L. reuteri can be obtained by employing these vectors and the final goal of our research, i.e., construction of expression-secretion vectors, can be reached as early as possible.

Indiana University-Purdue University Indianapolis (IUPUI)<br>The development of new methods to carry out gene transfer has many benefits to several fields, such as gene therapy, agriculture and animal health. The newly established lentiviral vector systems further increase the efficiency of gene transfer dramatically. Some studies have shown that lentiviral vector systems enhance efficiency over 10-fold higher than traditional pronuclear injection. However, the timing for lentiviral vector integration to occur remains unclear. Integrating in different stages of embryogenesis might lead to different integration patterns between tissues. Moreover, in our previous study we found that the vector copy number in transgenic sheep varied, some having one or more copies per cells while other animals having less than one copy per cell suggesting mosaicism. Here I hypothesized that injection of a lentiviral vector into a single cell embryo can lead to integration very early in embryogenesis but can also occur after several cell divisions. In this study, we focus on investigating integration sites in tissues developing from different germ layers as well as extraembryonic tissues to determine when integration occurs. In addition, we are also interested in insertional mutagenesis caused by viral sequence integration in or near gene regions. We utilize linear amplification-mediated polymerase chain reaction (LAM-PCR) and next- generation sequencing (NGS) technology to determine possible integration sites. In this study, we found the evidence based on a series of experiments to support my hypothesis, suggesting that integration event also happens after several cell divisions. For insertional mutagenesis analysis, the closest genes can be found according to integration sites, but they are likely too far away from the integration sites to be influenced. A well-annotated sheep genome database is needed for insertional mutagenesis analysis.