Controlling gene expression in mice with tetracycline: application in pigment cell research

Genetic manipulation techniques are widely used in mice to study the functions of genes. The most common strategy for assessing in vivo function involves making irreversible changes in the genome by homologous recombination. To complement this approach, a number of systems have been developed that allow specific and controlled expression of a gene. One of the more versatile and promising systems is based on the tetracycline (tet) responsive bacterial tetracycline repressor (TetR). In recent years, the tet system has proven to be a valuable method for understanding the function of genes involved in a number of physiological processes, including mouse models for human diseases such as cancer and neurological and pigment disorders. This review will highlight the power and elegance of the tet system by focusing on its utility in the study of two pigment cell-related biological problems, the pathogenesis of melanomas and melanocyte development in the embryo.     

Dual perfluorocarbon method to noninvasively monitor dissolved oxygen concentration in tissue engineered constructs in vitro and in vivo

Noninvasive in vivo monitoring of tissue implants provides important correlations between construct function and the observed physiologic effects. As oxygen is a key parameter affecting cell and tissue function, we established a monitoring method that utilizes ¹⁹F nuclear magnetic resonance (NMR) spectroscopy, with perfluorocarbons (PFCs) as oxygen concentration markers, to noninvasively monitor dissolved oxygen concentration (DO) in tissue engineered implants. Specifically, we developed a dual PFC method capable of simultaneously measuring DO within a tissue construct and its surrounding environment, as the latter varies among animals and with physiologic conditions. In vitro studies using an NMR‐compatible bioreactor demonstrated the feasibility of this method to monitor the DO within alginate beads containing metabolically active murine insulinoma βTC‐tet cells, relative to the DO in the culture medium, under perfusion and static conditions. The DO profiles obtained under static conditions were supported by mathematical simulations of the system. In vivo, the dual PFC method was successful in tracking the oxygenation state of entrapped βTC‐tet cells and the surrounding peritoneal DO over 16 days in normal mice. DO measurements correlated well with the extent of cell growth and host cell attachment examined postexplantation. The peritoneal oxygen environment was found to be variable and hypoxic, and significantly lower in the presence of metabolically active cells. The significance of the dual PFC system in providing critical DO measurements for entrapped cells and other tissue constructs, in vitro and in vivo, is discussed. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.     

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.     

Acquired tetracycline and/or macrolide-lincosamides-streptogramin resistance in anaerobes

In general bacterial antibiotic resistance is acquired on mobile elements such as plasmids, transposons and/or conjugative transposons. This is also true for many antibiotic resistant anaerobic species described in the literature. Of the 23 different tetracycline resistant efflux genes identified, tet(B), tet(K), tet(L), and tetA(P) have been found in anaerobic species and six of the ten tetracycline resistant genes coding for ribosomal protection proteins, tet(M), tet(O), tetB(P), tet(Q), tet(W), and tet(32), have been identified in anaerobes. There are now three enzymes which inactivate tetracycline, of which the tet(X) has been identified in Bacteroides though is not functional under anaerobic growth conditions. A similar situation exists with the genes conferring macrolide-lincosamide-streptogramin (MLS) resistance. Of the 26 rRNA methylase MLS resistant genes characterized, five genes; erm(B), erm(C), erm(F), erm(G), and erm(Q), have been identified in anaerobes. In contrast, no genes coding for MLS resistant efflux proteins or inactivating enzymes have been described in anaerobic species. This mini-review will summarize what is known about tetracycline and MLS resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes.     

Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression.

We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids. In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect. Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome. In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains. Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether. The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis. Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions. In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis. Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids. Differences between E. coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold. We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.     

Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance

We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). The assays were validated using known numbers of sample-derived tet gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log tet gene copies. New tet gene variants were also identified from cloned tet amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three tet gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of tet genes than the swine manures. The composts of swine manures had substantially reduced tet gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on tet gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of tet genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of tet genes, which should be useful for ecological studies of antibiotic resistance.     

Liposomal delivery of nucleic acids in vivo

Optimization of cationic liposomal complexes for in vivo applications and therapeutics is complex involving many distinct components. These components include nucleic acid purification, plasmid design, formulation of the delivery vehicle, administration route and schedule, dosing, detection of gene expression, and others. This review will focus on optimization of these components for use in a variety of in vivo applications. Use of improved liposome formulations for delivery in vivo is valuable for gene therapy and would avoid several problems associated with viral delivery. Delivery of nucleic acids using liposomes is promising as a safe and non-immunogenic approach to gene therapy. Furthermore, gene therapeutics composed of artificial reagents can be standardized and regulated as drugs rather than as biologics. Optimizing all components of the delivery system will allow broad use of liposomal complexes to treat or cure human diseases or disorders.     

Silencing and un-silencing of tetracycline-controlled genes in neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (P(tets)). We have discovered that stably integrated P(tet) becomes functionally silenced in the majority of neurons when it is inactive during development. P(tet) silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced P(tet) gene silencing, possibly by inducing promoter accessibility.     

SPICE: discovery of phenotype-determining component interplays

ABSTRACT: BACKGROUND: A latent behavior of a biological cell is complex. Deriving the underlying simplicity, or the fundamental rules governing this behavior has been the Holy Grail of systems biology. Data-driven prediction of the system components and their component interplays that are responsible for the target system's phenotype is a key and challenging step in this endeavor. RESULTS: The proposed approach, which we call System Phenotype-related Interplaying Components Enumerator (SPICE), iteratively enumerates statistically significant that are hypothesized (1) to play an important role in defining the specificity of the target system's phenotype(s); (2) to exhibit a functionally coherent behavior, namely, act in a coordinated manner to perform the phenotype-specific function; and (3) to improve the predictive skill of the system's phenotype(s) when used collectively in the ensemble of predictive models. When validated, SPICE effectively identified system components related to three target phenotypes: biohydrogen production, motility, and cancer. Manual results curation agreed with the known phenotype-related system components reported in literature. Additionally, using the identified system components as discriminatory features improved the prediction accuracy by 10% on the phenotype-classification task when compared to a number of state-of-the-art methods applied to eight benchmark microarray data sets. CONCLUSION: Conclusion: We formulate a problem--- enumeration of phenotype-determining system component interplays---and propose an effective methodology (SPICE) to address this problem. SPICE improved identification of cancer-related groups of genes from various microarray data sets and detected groups of genes associated with microbial biohydrogen production and motility, many of which were reported in literature. SPICE also improved the predictive skill of the system's phenotype determination compared to individual classifiers and/or other ensemble methods, such as bagging, boosting, random forest, nearest shrunken centroid, and random forest variable selection method. All the supporting data and software can be found in the supplemental files.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet(B) and tet(M) to the recipient. Both discriminatory PCR and sequence analysis showed that tet(M) genes of Vibrio spp. appear to be a single allele containing a specific region of tet(M) in Tn1545. However, erm(B) and aphA3, known to be linked to Tn1545-like genes, were not detected in Tc-resistant Vibrio spp., even in 9 strains resistant to erythromycin. In analysis to examine the relative position of tet(B) and tet(M), it was shown that tet(M) was present at the 3'-end of the insertion sequence IS10 of Tn10 carrying tet(B). At the junctional region between Tn10 and tet(M), we found a 14 bp sequence of unknown function and the deletion of regulatory sequences reported to be needed for tet(M) expression in conjugative transposons. This is the first report of the simultaneous presence of tet(B) and tet(M), and of the tet(M) gene being linked to the 3'-end of Tn10 in Tc-resistant Vibrio spp. in Korea.     

Detection of a common and persistent tet(L)-carrying plasmid in chicken-waste-impacted farm soil

The connection between farm-generated animal waste and the dissemination of antibiotic resistance in soil microbial communities, via mobile genetic elements, remains obscure. In this study, electromagnetic induction (EMI) surveying of a broiler chicken farm assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, a disparity existed between the two sites with regard to soil pH, tetracycline resistance (Tc(r)) levels among culturable soil bacteria, and the incidence and prevalence of several tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a relatively pristine regional forest. When the farm was in operation, tet(L), tet(M), tet(O), erm(A), erm(B), and erm(C) genes were detected in the waste-affected soil. Two years after all waste was removed from the farm, tet(L), tet(M), tet(O), and erm(C) genes were still detected. The abundances of tet(L), tet(O), and erm(B) were measured using quantitative PCR, and the copy numbers of each were normalized to eubacterial 16S rRNA gene copy numbers. tet(L) was the most prevalent gene, whereas tet(O) was the most persistent, although all declined over the 2-year period. A mobilizable plasmid carrying tet(L) was identified in seven of 14 Tc(r) soil isolates. The plasmid's hosts were identified as species of Bhargavaea, Sporosarcina, and Bacillus. The plasmid's mobilization (mob) gene was quantified to estimate its prevalence in the soil, and the ratio of tet(L) to mob was shown to have changed from 34:1 to 1:1 over the 2-year sampling period.     

Variability of Inducible Expression across the Hematopoietic System of Tetracycline Transactivator Transgenic Mice

The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.     

Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria

Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H(+) antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.     

Coadaptation and malaria control

Malaria emerges from a disequilibrium of the system 'human-plasmodium-mosquito' (HPM). If the equilibrium is maintained, malaria does not ensue and the result is asymptomatic plasmodium infection. The relationships among the components of the system involve coadaptive linkages that lead to equilibrium. A vast body of evidence supports this assumption, including the strategies involved in the relationships between plasmodium and human and mosquito immune systems, and the emergence of resistance of plasmodia to antimalarial drugs and of mosquitoes to insecticides. Coadaptive strategies for malaria control are based on the following principles: (1) the system HPM is composed of three highly complex and dynamic components, whose interplay involves coadaptive linkages that tend to maintain the equilibrium of the system; (2) human and mosquito immune systems play a central role in the coadaptive interplay with plasmodium, and hence, in the maintenance of the system's equilibrium; the under- or overfunction of human immune system may result in malaria and influence its severity; (3) coadaptation depends on genetic and epigenetic phenomena occurring at the interfaces of the components of the system, and may involve exchange of infectrons (genes or gene fragments) between the partners; (4) plasmodia and mosquitoes have been submitted to selective pressures, leading to adaptation, for an extremely long while and are, therefore, endowed with the capacity to circumvent both natural (immunity) and artificial (drugs, insecticides, vaccines) measures aiming at destroying them; (5) since malaria represents disequilibrium of the system HPM, its control should aim at maintaining or restoring this equilibrium; (6) the disequilibrium of integrated systems involves the disequilibrium of their components, therefore the maintenance or restoration of the system's equilibrium depend on the adoption of integrated and coordinated measures acting on all components, that means, panadaptive strategies. Coadaptive strategies for malaria control should consider that: (1) host immune response has to be induced, since without it, no coadaptation is attained; (2) the immune response has to be sustained and efficient enough to avoid plasmodium overgrowth; (3) the immune response should not destroy all parasites; (4) the immune response has to be well controlled in order to not harm the host. These conditions are mostly influenced by antimalarial drugs, and should also be taken into account for the development of coadaptive malaria vaccines.     

The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant.

Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed.