Target Cassette Research Articles

Evaluation of Recombinant Antibody Production Efficiency in CHO Cells with Sleeping Beauty Transposon Vector System

Chinese hamster ovary (CHO) mammalian cell lines are widely used as cell platforms in biopharmaceutical productions. Different transfection systems are employed for the integration of the target gene cassette into the cell genome and have limitations, such as (i) the integration region in the genome, (ii) the size of the target cassette, and (iii) long selection periods for stable expression. Transposon systems can be utilized to overcome the limitations mentioned in the efficient production of commercially significant recombinant proteins. This study aims to demonstrate the differences in production potential and selection periods by using a specially designed vector system for random genome integration in CHODG44 DHFR -/- cells and the Sleeping Beauty (SB) transposon system. In this context, the optimal transfer ratio between the donor and the helper plasmid was determined for the most efficient co-transfection in the SB transposon system. According to the results, the pools obtained using the SB transposon system had titers ranging from 1300 to 2600 mg/L in 13-day fed-batch studies, while the pool obtained using the random transfer system had a titer of 0.056 mg/L. Additionally, stable cell pools obtained using the transposon system underwent selection in a short period of 52 days, compared to over 100 days for the pool obtained through random transfer. Considering all these results together, it is demonstrated that stable CHO pools obtained using the optimal SB transposon system can achieve high-efficiency monoclonal antibody production in a short period, making it an optimal production platform in the biopharmaceutical field.

Insect High Five™ cell line development using site-specific flipase recombination technology.

Insect Trichoplusia ni High Five™ (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e., iCherry, as an intracellular protein model) or two proteins (i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.

Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies.

PurposeThe purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases.MethodsCorneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12.ResultsThe Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2).ConclusionsThis novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.

Design of an Interferon-Resistant Oncolytic HSV-1 Incorporating Redundant Safety Modalities for Improved Tolerability

Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.

Enhanced Production of Recombinant Alcohol Dehydrogenase Using the Genetically Engineered Escherichia coli Strain that Heterologously Expresses Carrot Heat Shock Protein 70.

Escherichia coli (E. coli) has been widely used as a host organism for producing recombinant proteins such as biocatalysts, antibody fragments, and therapeutic hormones. To enhance recombinant protein production, many E. coli strains have been genetically engineered on practical purposes. In this study, we developed the engineered E. coli strain expressing Heat shock protein 70, DcHsp70, from carrot (Daucus carota L.). The DNA construct for DcHsp70 expression, Lipoprotein promoter-DcHsp70 gene-Flippase recognition target cassette, which is flanked by the insertion site yddE pseudogene sequences, was generated by overlap PCR and inserted into the E. coli genome by lambda Red-mediated homologous recombination. To examine if the engineered E. coli cells can effectively produce recombinant proteins, the alcohol dehydrogenase (ADH) gene from a thermophile, Geobacillus stearothermophilus, was cloned into a pET11a expression vector and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. Compared to wild type, the genetically engineered E. coli expressing DcHsp70 exhibited up to approximately 11-fold higher production of his-tagged ADH, mostly in soluble forms. The his-ADH protein that was purified from the engineered cells exhibited the enzyme activity. The genetically engineered E. coli developed in this study can be useful for the efficient production of recombinant proteins, such as recombinant ADH.

Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii.

The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application.IMPORTANCEAcinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

Expression of the VP1 protein of FMDV integrated chromosomally with mutant Listeria monocytogenes strain induced both humoral and cellular immune responses.

Live vector-based vaccine is a modern approach to overcome the drawbacks of inactivated foot-and-mouth disease (FMD) vaccines such as improper inactivation during manufacture. Listeria monocytogenes (LM), an intracellular microorganism with immune-stimulatory properties, is appropriate to be utilized as a live bacterial vaccine vector. FMDV-VP1 protein has the capability to induce both cellular and humoral immune responses since it is considered the most immunogenic part of FMDV capsid and has the most of antigenic sites for viral neutralization. The codon-optimized vp1 gene was ligated to the integrative pCW702 plasmid to construct the target cassette. The antigen cassette was integrated successfully into the chromosome of mutant LM strain via homologous recombination for more stability to generate a candidate vaccine strain LM△actAplcB-vp1. Safety evaluation of recombinant LM△actAplcB-vp1 revealed it could be eliminated from the internal organs within 3days as a safe candidate vaccine. Mice groups were immunized I.V. twice with the recombinant LM△actAplcB-vp1 at an interval of 2weeks. Antigen-specific IgG antibodies and the level of CD4+- and CD8+-specific secreted cytokines were estimated to evaluate the immunogenicity of the candidate vaccine. The rapid onset immune response was detected, strong IgG humoral immune response within 14days post immunization and augmented again after the booster dose. Cellular immunity data after 9days post the prime dose indicated elevation in CD4+ and CD8+ secreted cytokine level with another elevation after the booster dose. This is the first report to explain the ability of attenuated mutant LM to be a promising live vector for FMDV vaccine.

DNA Preparation from Schizosaccharomyces pombe.

This protocol includes two methods for genomic DNA preparation from Schizosaccharomyces pombe The first is a quick method for preparation of DNA suitable for polymerase chain reaction (PCR) genotyping. The second, longer method yields high-quality DNA that can be used for amplification of targeting cassettes.

A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

Development of Transgenic Escherichia coli with Improved Viability by Heterologous Expression of a Heat Shock Protein Gene from Carrot (Daucus carota L.)

A small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the Escherichia coli chromosome by RecE/RecT-based homologous recombination to increase cell viability during industrial fermentation, which frequently encounters adverse growth conditions. DNA construct “lipoprotein (Lpp) gene promoter—Hsp17.7 gene—flippase recombination target (Frt) cassette” flanked by the sequences of the insertion site of the E. coli chromosome (yddE pseudogene) was generated by polymerase chain reaction (PCR). The transformed E. coli cell lines that heterologously expressed Hsp17.7 exhibited shorter lag phase, compared with control cell line under normal (37 °C), heat (45 °C), and antifoam conditions. Cell viability was higher in the transformed cell lines in the heat (50 °C, up to 2-fold) and cold (2 °C, up to 1.7-fold) conditions. The soluble protein levels were also higher in the transformed E. coli cell lines by up to 20%, compared with control cell line in both stress conditions. The stress-tolerant transgenic cell lines developed in this study can contribute to more efficient and cost saving industrial cultivation of E. coli, which is most frequently used for recombinant protein production.

Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells.

Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines.

358 EXCESS EXPRESSION OF 11β-HSD1 CONTRIBUTES TO LETHALITY THROUGH DYSFUNCTION IN ENERGY BALANCE BETWEEN ANABOLIC PROCESS AND ENERGY RECOVERY PROCESS

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) effectively amplifies glucocorticoid action in liver, adipose tissue, and brain. This enzyme converts the inactive 11-keto form into a glucocorticoid (e.g. cortisol). Glucocorticoids (GC) are steroid hormones found in the body and produced by the adrenal cortex, the outer layer of the adrenal glands. Glucocorticoids are lipophilic and readily access their intracellular receptors. Glucocorticoids regulate carbohydrate, fat, and protein metabolism. In a previous study, we constructed a vector composed of 2 parts: the 11β-HSD1 expression cassette and the selection cassette containing EGFP and Neo resistant genes. Porcine fibroblasts overexpressing 11β-HSD1 under the control of adipose tissue-specific aP2 promoter were established and used in somatic cell nuclear transfer (SCNT). Somatic cells from the resulting stillborn transgenic piglets were used in a second round of SCNT. Non-obese, transgenic piglets overexpressing 11β-HSD1 were obtained and were identified through PCR-based methods using specific primers for the targeting cassettes from the genomic DNA of piglets. Six live piglets, 1 stillborn piglet, and 3 mummies were born. Integration of target gene into the genomic DNA was confirmed for all of them. However, all 6 live piglets died within 1 month. All of the piglets had displayed hypoglycemia. Increased expression of 11β-HSD1 in metabolic tissues induced up-regulation of gluconeogenesis-related genes (G6PT, G6Pase, PEPCK, HNF4a, FOXO1) in liver and kidney, and showed up-regulation of lipogenesis-related genes (SREBP1c, FASN, DGAT, ACC, SCD) in muscle. The AMPK and SIRT signalling, which controls energy balance and mitochondrial biogenesis, was also stimulated. We propose that overexpression of 11β-HSD1 evokes the excess production and action of glucocorticoid or its receptors, and activates gluconeogenic and lipogenic pathways. For this reason, AMPK and SIRT1 signalling was induced. Also, in compensation of energy loss by anabolic processes, the expression of mitochondrial biogenesis-related genes was increased. Finally, the constitutive expression of 11β-HSD1 might continuously activate complementary energy-gaining processes, and these problems could develop into more fatal diseases that resulted in the piglets' death.

Captured Segment Exchange: A Strategy for Custom Engineering Large Genomic Regions inDrosophila melanogaster

Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition-site insertions can be "captured" as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50-kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition-site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.

Abstract 5654: Novel tissue-targeted virogeneimaging system for squamous cell carcinoma therapy expressing cell cycle regulator cdk2ap1

Abstract Oral squamous cell carcinoma (OSCC) affects about 30,000 Americans each year, and although the overall prognosis has improved in recent years for localized carcinomas, metastases can decrease survival rates by half. For tongue and palate lesions, surgery is usually the initial treatment, but metastases aggressively develop at sites where conventional therapies are ineffective. Therefore, our goal has been to develop more effective therapeutic modalities for the disease utilizing expression of cell cycle arrest and apoptosis-modulating genes. Our efforts have focused on gaining understanding of the biology and therapeutic potential of p12cdk2ap1 (p12), an S-phase growth suppressor and proapoptotic gene commonly downregulated in OSCC. In vivo, we have showed that expressing p12 using a nonviral vector can result in effective reduction in tumor size and a dramatically altered tumor phenotype. Moreover, we have showed that p12 can have cell growth inhibitory effects in a cell-autonomous as well as non-cell autonomous manner, rendering expression of this cell cycle regulator a very promising new gene therapy strategy. Therefore, we hypothesized that this growth regulator molecule will comprise a highly efficient antiumor gene therapy for OSCC. We have developed a novel viral delivery vector system with high activity and high specificity expression in oral tumor cells. This Adenovirus (Ad) utilizes a two-step transcriptional amplification system driven by the SCCA2 promoter and a bidirectional target cassette containing the sequences for the p12 and ether firefly luciferase (Luc) or thymidine kinase (TK) imaging genes. We constructed and are characterizing novel configurations of these dE1dE3 Ad-SCCA2-p12/Luc or p12/TK bidirectional vectors for gene therapy and virogeneimaging (VGI) applications. We are currently testing the specificity of these vectors and their effectiveness in tumor growth inhibition at either floor of mouth, orthotopic, or distant sites following intratumoral or systemic administration. We propose VGI can be adapted to a nonviral system as well and may hold promise not only as a novel SCC-specific gene therapy but perhaps as a molecular imaging detection agent for tumor cells that have metastasized to distant and thus difficult to treat sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5654. doi:1538-7445.AM2012-5654

Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

Systematic Targeted Integration to Study Albumin Gene Control Elements

To study transcriptional regulation by distant enhancers, we devised a system of easilymodified reporter plasmids for integration into single-copy targeting cassettes in clones of HuH7, a human hepatocellular carcinoma. The plasmid constructs tested transcriptional function of a 35-kb region that contained the rat albumin gene and its upstream flanking region. Expression of integrants was analyzed in two orientations, and compared to transient expression of non-integrated plasmids. Enhancers were studied in their natural positions relative to the promoter and localizedby deletion. All constructs were also analyzed by transient transfection assays. In addition to the known albumin gene enhancer (E1 at −10 kb), we demonstrated two new enhancers, E2 at −13, and E4 at +1.2 kb. All three enhancers functioned in both transient assays and integrated constructs. However, chromosomal integration demonstrated several differences from transient expression. For example, analysis of E2 showed that enhancer function within the chromosome required a larger gene region than in transient assays. Another conserved region, E3 at −0.7 kb, functioned as an enhancer in transient assays but inhibited the function of E1 and E2 when chromosomally integrated. The enhancers did not show additive or synergistic behavior,an effect consistent with competition for the promoter or inhibitory interactions among enhancers. Growth arrest by serum starvation strongly stimulated the function of some integrated enhancers, consistent with the expected disruption of enhancer-promoter looping during the cell cycle.

A method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy

Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains.

Abstract 5414: Novel tissue-targeted adenoviral vector for squamous cell carcinoma therapy expressing cell cycle regulator cdk2ap1 and molecular imaging genes

Abstract Oral squamous cell carcinoma (OSCC) affects about 30,000 Americans each year, and although the overall prognosis has improved in recent years for localized carcinomas, metastases can decrease survival rates by half. For tongue and palate lesions, surgery is usually the initial treatment, but metastases aggressively develop at sites where conventional therapies are ineffective. Therefore, our goal has been to develop more effective therapeutic modalities for the disease utilizing expression of cell cycle arrest and apoptosis-modulating genes. Our efforts have focused on gaining understanding of the biology and therapeutic potential of p12cdk2ap1 (p12), an S-phase growth suppressor and proapoptotic gene commonly downregulated in OSCC. In vivo, we have showed that expressing p12 using a nonviral vector can result in effective reduction in tumor size and a dramatically altered tumor phenotype. Moreover, we have showed that p12 can have cell growth inhibitory effects in a cell-autonomous as well as non-cell autonomous manner, rendering expression of this cell cycle regulator a very promising new gene therapy strategy. Therefore, we hypothesized that this growth regulator molecule will comprise a highly efficient antiumor gene therapy for OSCC. We have developed a novel viral delivery vector system with high activity and high specificity expression in oral tumor cells. This Adenovirus (Ad) utilizes a two-step transcriptional amplification system driven by the SCCA2 promoter and a bidirectional target cassette containing the sequences for the p12 and ether firefly luciferase (Luc) or thymidine kinase (TK) imaging genes. We constructed and are characterizing novel configurations of these dE1dE3 Ad-SCCA2-p12/Luc or p12/TK bidirectional vectors for gene therapy and molecular imaging applications. We are currently testing the specificity of these vectors and their effectiveness in tumor growth inhibition at either floor of mouth, orthotopic, or distant sites following intratumoral or systemic administration. We propose this new vector design may hold promise not only as a novel SCC-specific gene therapy but perhaps as a molecular imaging detection agent for tumor cells that have metastasized to distant and thus difficult to treat sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5414. doi:10.1158/1538-7445.AM2011-5414

Sulfolobus mutants, generated via PCR products, which lack putative enzymes of UV photoproduct repair.

In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens

Penicillium decumbens is an important industrial filamentous fungus and has been widely used in biorefinery due to its high production of cellulase and hemicellulase. However, molecular engineering has still rarely been applied for strain improvement in P. decumbens. It has been proven that gene targeting manipulation in many filamentous fungi is hampered by nonhomologous end-joining (NHEJ) pathway. To improve gene targeting efficiency in P. decumbens, the putative pku70 encoding the Ku70 homologue involved in the NHEJ pathway was identified and deleted. The Deltapku70 strain showed no apparent defect in vegetative growth, conidiation, and cellulase production, and displayed similar sensitivity to chemical agents of hygromycin B, ethyl methane sulfonate, and H2O2 at different concentrations compared with the wild-type strain. The effect of the absence of pku70 on gene targeting was tested by disruption of creA encoding a putative carbon catabolite repressor and xlnR encoding a putative transcriptional activator. Efficiency of gene targeting for both genes was 100% in the Deltapku70 strain, compared with the low efficiency in the wild-type recipient. Furthermore, the integration types for three single targeting cassettes and the cotransformation of two independent targeting cassettes were primarily investigated in P. decumbens. The highly efficient gene targeting system established in this study will open the way to large-scale functional genomic analysis in P. decumbens and contribute to the study of the mechanism of lignocellulose degradation by P. decumbens.