Plasmid Markers Research Articles

Tumor growth reduction and DNA transfer by cavitation-enhanced high-intensity focused ultrasound in vivo

The potential application of high-intensity focused ultrasound (US), HIFU, was investigated for nonthermal gene transfer and tumor ablation. Renal carcinoma (RENCA) tumors were implanted on the hind leg of BALB/c mice and injected with a marker plasmid. Optison® US contrast agent was also injected into the tumor (IT) or into the venous (IV) circulation. HIFU at 1.55 MHz was applied to the tumors with guidance from diagnostic US images. One test of transfection was also performed with lithotripter shock waves. In one set of exposures, tumor volume was followed for 4 days and a β-galactosidase marker plasmid was used for localization of transfected cells. A second set of exposures employed a luciferase marker plasmid for assessing overall transfection after 2 days. Use of 100-ms bursts at 8-MPa peak rarefactional pressure amplitude stopped tumor growth during the 4-day period, compared to a 2.8-fold growth in shams and yielded luciferase expression 34-fold greater than in shams. Longer bursts or higher pressure amplitudes led to decreases in tumor growth, but did not yield increases in transfection. The HIFU results were similar to those of shock waves for cavitation enhanced by IT Optison®. These results should aid in optimizing the application of HIFU for nonthermal tumor treatment.

Selection of novel eukaryotic DNA polymerases by mutagenesis and genetic complementation of yeast.

AbstractDNA-directed DNA polymerases have been broadly classified into seven families based on their sequence homology (1). It is surprising to learn that enzymes such as DNA polymerases, which carry out pivotal role during DNA replication, repair, and recombination, are poorly conserved amongst different families, but within a given family, all the members are highly conserved. These observations have profound implications and suggest that DNA polymerases have been plastic during evolution, but can tolerate multiple mutations (2). The mutability of DNA polymerases has been utilized extensively in our studies and has shed light on structure-function relationships of each domain. Any single amino acid residue or the entire domain can be randomly mutagenized and the active mutants can be selected by genetic complementation. Here we describe the complementation of Saccharomyces cerevisiae Pol3 (Pol δ) by utilizing a common technique in yeast genetics known as “plasmid shuffling,” where the wild-type copy of the Pol3 present in a Ura3 selective marker plasmid is exchanged or genetically complemented for in vitro mutated version(s) of Pol3 in the domain-of-interest. Since Pol3p is essential for viability of yeast, only those mutants that genetically complement the loss of wild-type Pol3p survive. KeywordsYeast StrainMultiple Cloning SiteGenetic ComplementationPlasmid RescueUra3 MarkerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Gap repair transformation in fission yeast to exchange plasmid-selectable markers.

Gap repair transformation in fission yeast to exchange plasmid-selectable markers.

Inducible Gene Expression Using an Autoregulatory, Tetracycline‐Controlled System

The protocols in this unit describe the transfection of adherent cells and the testing of resultant clones for inducible transactivator or target gene protein expression. Stably transfected fibroblast cell lines expressing transactivator and target gene(s) can be derived by first cotransfecting pTet-tTAk and a plasmid encoding a selectable marker and obtaining stable lines with inducible transactivator expression. These lines are subsequently stably cotransfected with plasmids encoding the target gene(s) and a second selectable marker. The procedure may also be used to cotransfect pTet-tTAk with the target gene-encoding plasmid(s) and a single selectable marker plasmid. A support protocol describes methods to test stably transfected cell lines for inducible gene expression, for transient transfection and induction of tet-regulated plasmids, and for detection of the tTAk gene in cells (or transgenic mice).

Lithotripter shock waves with cavitation nucleation agents produce tumor growth reduction and gene transfer in vivo

Cavitation nucleation agents (CNA) can greatly enhance DNA transfer and cell killing for therapeutically useful applications of nonthermal bioeffects of ultrasound (US). Renal carcinoma (RENCA) tumor cells were implanted and grown to about 400 μL tumor volumes on the hind legs of syngeneic Balb/c mice. Before treatment, mice were anesthetized, the tumor region was shaved and depilated, and a DNA plasmid coding for marker proteins was injected into the tumor. Two sets of tests were completed: the first set involved measurement of tumor growth for 4 days and use of a β-galactosidase marker plasmid for localization of transfection, and the second set involved 2 days of growth and use of a luciferase marker plasmid for assessing overall protein expression. Either saline, Optison® US contrast agent, a vaporizing perfluoropentane droplet suspension (SDS) or air bubble was also injected intratumorally at 10% of tumor volume as a CNA. In some tests, droplets or contrast agent were injected IV. Shock waves (SW) were generated from a spark-gap lithotripter at 7.4 MPa peak negative pressure amplitude. For sham exposure, tumor volume increased by a factor of 3.6 in 4 days. With 500-SW treatment, all the CNA reduced 4-day tumor growth about the same amount (to factors of 1.2 to 1.9). Marker gene expression was generally localized to the region around the needle injection path. All the agents, except saline, produced statistically significant increases of 11.8- to 14.6-fold in luciferase expression after 2 days, relative to sham exposure. IV injection of Optison® or droplet nucleation agents before SW treatment reduced tumor growth to factors of 1.0 and 0.7, but did not increase transfection. These results demonstrate the efficacy of CNA in vivo and should lead to improved strategies for simultaneous SW tumor ablation and cancer gene therapy. (E-mail: douglm@umich.edu)

Characterization of Borrelia burgdorferi isolated from erythema migrans lesions: interrelationship of three molecular typing methods.

Genetic diversity among Borrelia burgdorferi isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content (P < 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.

A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis.

A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated. Cryptic plasmids were mixed with an erythromycin resistance (Eryr) marker plasmid and introduced into a recipient strain by electroporation, followed by plasmid extraction of erythromycin-resistant transformants. By optimizing the ratio between the marker plasmid and the cryptic plasmids, an average of 20% cotransformants was obtained, including combinations of more than one cryptic plasmid. The marker plasmid pSA3 was easily eliminated from the cotransformed cells by subculture without selective pressure. This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids. The method facilitates the isolation of cryptic plasmids, helps in assigning functions to unknown plasmids and allows construction of food-grade lactococcal strains with new combinations of wild-type plasmids.

Development of genetic markers in cyanobacteria and stability of genetically marked strains in soil

Reporter marker GUS (β-glucuronidase gene from Escherichia coli) and luc operon from the American firefly were introduced into cyanobacteria and the stability of these markers in soil was examined. To transfer the integrational vector into cyanobacteria, the genomic DNA library of Synechocystis sp. or Anabaena cylindrica maintained in pBR 322, pCY 100 and pCY 101 were transformed with HB 101 containing pRL 528 and selected for Cmr and Ampr. These clones of HB 101 containing pRL 528 and the vectors carrying different cyanobacterial chromosomal DNA fragments were used for triparental mating with HB 101 [pRK 2013/pRK 2073] and cyanobacteria. The frequency of transconjugants for integrational vectors was between 2.1 × 10−5 and 4.0 × 10−4. The transfer frequency of RSF 1010 based vectors (pDSK 519 and pCY 106) was 1.0–4.5 × 10−4 in Synechocystis sp. whereas A. cylindrica failed to maintain these vectors. Low frequency transfer (2.0–2.3 × 10−6) of RK 2 based vectors pVK 100 and pCY 104 was observed in A. cylindrica but these were unable to replicate in Synechocystis sp. The vectors in general were stable at least by 74.9% for 60 days of incubation in BG-11 medium. The markers were less stable in A. cylindrica (74.9–84.2%) compared to Synechocystis sp. (80.1–88.8%) at 60 days of incubation. Integrational vectors were almost 85% stable in both the strains. The RK 2 derivative of pCY 104 was less stable in A. cylindrica (74.9–77.3%) than the RSF 1010-based vector pCY 106 in Synechocystis sp. (80.1–81.0%). A maximum of 64.7% of the markers were lost in soil. The chromosomal markers through integrational vectors were found to be highly stable and 68.2–72.7% of these markers were retained in cyanobacteria at 60 days of incubation. Plasmid markers were less stable, with a loss of 64.7–48.7% at the end of the experiment. In A. cylindrica 58–65% of the RK 2 vector was lost whereas in Synechocystis sp. 49–61% of RSF 1010 was lost at 60 days of incubation.

Direct inhibition of caspase 3 is dispensable for the anti-apoptotic activity of XIAP.

XIAP is a mammalian inhibitor of apoptosis protein (IAP). To determine residues within the second baculoviral IAP repeat (BIR2) required for inhibition of caspase 3, we screened a library of BIR2 mutants for loss of the ability to inhibit caspase 3 toxicity in the yeast Schizosaccharomyces pombe. Four of the mutations, not predicted to affect the structure of the BIR fold, clustered together on the N-terminal region that flanks BIR2, suggesting that this is a site of interaction with caspase 3. Introduction of these mutations into full-length XIAP reduced caspase 3 inhibitory activity up to 500-fold, but did not affect its ability to inhibit caspase 9 or interact with the IAP antagonist DIABLO. Furthermore, these mutants retained full ability to inhibit apoptosis in transfected cells, demonstrating that although XIAP is able to inhibit caspase 3, this activity is dispensable for inhibition of apoptosis by XIAP in vivo.

Safety and Pharmacokinetics of Naked Plasmid DNA in the Skin: Studies on Dissemination and Ectopic Expression

Gene therapy using naked DNA injected into muscle and skin is increasingly being used for vaccination and treatment purposes. Favorably, naked plasmid DNA does not exhibit the various limitations inherent to viral vectors, such as the elicitation of adverse immune responses and the risk of insertional mutagenesis. In order to assess the distribution and safety of naked plasmid DNA in a relevant animal model, we analyzed if intracutaneously injected plasmid DNA was transported to other organs and if ectopic expression occurred. When a "superdose" of a marker plasmid was injected intradermally, most organs were found transiently to contain the plasmid DNA for several days, whereas integration into the host genome was not detected. With the exception of ovary, however, mRNA expression only occurred in the skin, regional lymph nodes, and muscular tissues. From a safety standpoint, skin gene therapy with naked plasmid DNA can be considered safe due to the rapid biodegradation of plasmid DNA and the exclusive and transient expression of foreign genes in tissues known to take up DNA.

Construction of a GFP-BAR plasmid and its use for switchgrass transformation.

A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l-1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T1 plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene.

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae.

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.

Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Higher diversity of Rhizobium leguminosarum biovar viciae populations in arable soils than in grass soils.

The bacterial genetic diversity after long-term arable cultivation was compared with that under permanent grassland using replicated paired contrasts. Pea-nodulating Rhizobium leguminosarum populations were sampled from pairs of arable and grass sites at four locations in Yorkshire, United Kingdom. Isolates were characterized using both chromosomal (16S-23S ribosomal DNA internal transcribed spacer PCR-restriction fragment length polymorphism) and plasmid (group-specific repC PCR amplification) markers. The diversities of chromosomal types, repC profiles, and combined genotypes were calculated using richness in types (adjusted to equal sample sizes by rarefaction), Shannon-Wiener index, and Simpson's index. The relative differences in diversity within each pair of sites were similar for all three diversity measures. Chromosomal types, repC profiles, and combined genotypes were each more diverse in arable soils than in grass soils at two of the four locations. The other comparisons showed no significant differences. We conclude that rhizobial diversity can be affected by differences between these two management regimens. Multiple regression analyses indicated that lower diversity was associated with high potential nitrogen and phosphate levels or with acidity.

A set of Hansenula polymorpha integrative vectors to construct lacZ fusions

A set of YEp Saccharomyces cerevisiae-based, integrative Hansenula polymorpha plasmids was constructed to express lacZ gene under yeast gene promoters. The HpLEU2 and HpURA3 genes were used both as markers and to target the integration of plasmids into the corresponding H. polymorpha genome locus. The frequency of transformation reached with these plasmids linearised either in HpLEU2 or HpURA3 was around 100 transformants per microgram of plasmid DNA; in all transformants checked by Southern blotting the plasmid was integrated into the genome locus corresponding to the gene plasmid marker. PCR showed that about 50% of the transformants contained more than one plasmid copy per genome. Experiments carried out using the developed plasmids to determine the strength of the gene promoters involved in nitrate assimilation in H. polymorpha revealed that, in the presence of nitrate, the nitrate reductase gene promoter (YNR1) was the strongest, followed by nitrite reductase (YNI1) and nitrate transporter (YNT1).

A positive selection for plasmid loss in Saccharomyces cerevisiae using galactose-inducible growth inhibitory sequences.

Counter-selections for the loss of introduced plasmid sequences are useful for gene manipulations in yeast. We have used GAL10 promoter-mediated overexpression of GIN sequences, which inhibit the growth of cells, to develop a novel counter-selection system. Yeast cells carrying a GIN sequence grow normally on glucose medium but are unable to grow on galactose medium, whereas derivatives that have lost the GIN sequence are able to grow in the presence of galactose. We constructed autonomously replicating, integrating, and disruption plasmids carrying GIN sequences and tested their use to select for loss of the plasmid. The results showed that the GIN sequences provide a selection for efficient loss of plasmids or integrated constructs from yeast during growth on galactose medium, indicating that this system can be used for plasmid shuffling, gene replacements and marker gene recycling. This counter-selection system has wide application, because any Gal+ strain and a wide variety of marker genes can be used. In addition, counter-selection systems using growth-inhibitory sequences should be applicable to other yeasts and possibly to other organisms.

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T2 plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.

Simple and Fast Method to Test the Receptor for Advanced Glycosylated Endproducts (RAGE) For its Tumor Suppressive Potential Using the Tet-On System

The Tet gene expression system, that allows tightly controlled gene expression in response to doxycycline, was applied to analyze the influence of the receptor for advanced glycosylation endproducts (RAGE) on the growth of 293 cells in semi-solid medium. Establishing a Tet-On gene expression system involves two consecutive stable transfections. Here, we describe an alternative procedure to obtain a Tet-On gene expression system in a single transfection step for the use in tumor biology. The plasmids necessary for the regulated expression of RAGE together with the selectable marker plasmid were cotransfected in a molar ratio of 6:1. After aminoglycoside selection, 29 clones were analyzed using PCR revealing 8 colonies to be double stably transformed. Subsequent Western blot analysis showed inducible expression in 7 cell lines. Applying the one step protocol, the entire Tet-On expression system could be completed in half of the time required for the original two step method. The generated 293 double stable cells were used in the clonogenic assay for the testing of the tumor suppressive potential of RAGE.

In vitro construction of pseudovirions of human papillomavirus type 16: incorporation of plasmid DNA into reassembled L1/L2 capsids.

Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a beta-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced beta-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.

Complementation of a Replication-Defective Mutant of Equine Herpesvirus Type 1 by a Cell Line Expressing the Immediate-Early Protein

Equine herpesvirus type 1 (EHV-1) possesses a sole, diploid immediate-early (IE) gene that encodes a major regulatory protein of 1487 amino acids capable of modulating expression of both early and late EHV-1 promoters and capable oftrans-repressing its own promoter. In this study, a rabbit kidney cell line (IE13.1) that constitutively expresses the EHV-1 IE protein was generated by cotransfection of rabbit kidney (RK-13) cells with the viral IE gene and a neomycin resistance marker. The IE protein expressed by this cell line was shown (1) to be expressed by and to localize to the nucleus of virtually all cells as demonstrated by indirect immunofluorescence, (2) to be the full-size IE polypeptide as judged by Western immunoblot analyses with an anti-IE protein-specific antibody, and (3) to be functional as shown by the transactivation of two representative EHV-1 early promoters linked to the chloramphenicol acetyltransferase reporter gene in transient transfection assays. The IE13.1 cell line was able to complement a recombinant virus in which both copies of the IE gene were replaced by insertion of theEscherichia coli lacZgene. This IE deletion mutant, designated KyAΔIE, was not able to replicate in equine, rabbit, or mouse cells but was capable of replication in the IE13.1 cells that provided the IE protein intrans. Rescue of the KyAΔIE virus was achieved by recombination with a marker plasmid that harbors the wild-type IE gene, and the rescued virus (KyAΔIER) was able to grow on noncomplementary cells. Overall, these results offer direct evidence that the IE gene is essential for EHV-1 replication and provide reagents useful for the analysis of IE protein function.