Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25–30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.     

Monitoring immediate-early gene expression through firefly luciferase imaging of HRS/J hairless mice

Background  

Gene promoters fused to the firefly luciferase gene (luc) are useful for examining gene regulation in live transgenic mice and they provide unique views of functioning organs. The dynamics of gene expression in cells and tissues expressing luciferase can be observed by imaging this enzyme's bioluminescent oxidation of luciferin. Neural pathways involved in specific behaviors have been identified by localizing expression of immediate-early genes such as c-fos. A transgenic mouse line with luc controlled by the human c-fos promoter (fos::luc) has enabled gene expression imaging in brain slice cultures. To optimize imaging of immediate-early gene expression throughout intact mice, the present study examined fos::luc mice and a second transgenic mouse containing luc controlled by the human cytomegalovirus immediate-early gene 1 promoter and enhancer (CMV::luc). Because skin pigments and hair can significantly scatter light from underlying structures, the two transgenic lines were crossed with a hairless albino mouse (HRS/J) to explore which deep structures could be imaged. Furthermore, live anesthetized mice were compared with overdosed mice.     

The Human Obesity Gene Map: The 2002 Update

This is the ninth update of the human obesity gene map, incorporating published results through October 2002 and continuing the previous format. Evidence from single‐gene mutation obesity cases, Mendelian disorders exhibiting obesity as a clinical feature, quantitative trait loci (QTLs) from human genome‐wide scans and various animal crossbreeding experiments, and association and linkage studies with candidate genes and other markers is reviewed. For the first time, transgenic and knockout murine models exhibiting obesity as a phenotype are incorporated (N = 38). As of October 2002, 33 Mendelian syndromes relevant to human obesity have been mapped to a genomic region, and the causal genes or strong candidates have been identified for 23 of these syndromes. QTLs reported from animal models currently number 168; there are 68 human QTLs for obesity phenotypes from genome‐wide scans. Additionally, significant linkage peaks with candidate genes have been identified in targeted studies. Seven genomic regions harbor QTLs replicated among two to five studies. Attempts to relate DNA sequence variation in specific genes to obesity phenotypes continue to grow, with 222 studies reporting positive associations with 71 candidate genes. Fifteen such candidate genes are supported by at least five positive studies. The obesity gene map shows putative loci on all chromosomes except Y. More than 300 genes, markers, and chromosomal regions have been associated or linked with human obesity phenotypes. The electronic version of the map with links to useful sites can be found at http:obesitygene.pbrc.edu .     

Transgenic hybrid aspen overexpressing the Atwbc19 gene encoding an ATP-binding cassette transporter confers resistance to four aminoglycoside antibiotics

Antibiotic-resistance genes of bacterial origin are invaluable markers for plant genetic engineering. However, these genes are feared to pose possible risk to human health by horizontal gene transfer from transgenic plants to bacteria, potentially resulting in antibiotic-resistant pathogenic bacteria; this is a considerable regulatory concern in some countries. The Atwbc19 gene, encoding an Arabidopsis thaliana ATP-binding cassette transporter, has been reported to confer resistance to kanamycin specifically as an alternative to bacterial antibiotic-resistance genes. In this report, we transformed hybrid aspen (Populus canescens × P. grandidentata) with the Atwbc19 gene. Unlike Atwbc19-transgenic tobacco that was only resistant to kanamycin, the transgenic Populus plants also showed resistance to three other aminoglycoside antibiotics (neomycin, geneticin, and paromomycin) at comparable levels to plants containing a CaMV35S-nptII cassette. Although it is unknown why the transgenic Populus with the Atwbc19 gene is resistant to all aminoglycoside antibiotics tested, the broad utility of the Atwbc19 gene as a reporter gene is confirmed here in a second dicot species. Because the Atwbc19 gene is plant-ubiquitous, it might serve as an alternative selectable marker to current bacterial antibiotic-resistance marker genes and alleviate the potential risk for horizontal transfer of bacterial-resistance genes in transgenic plants.     

How Epigenomics Contributes to the Understanding of Gene Regulation in Toxoplasma gondii1

ABSTRACT. How apicomplexan parasites regulate their gene expression is poorly understood. The complex life cycle of these parasites implies tight control of gene expression to orchestrate the appropriate expression pattern at the right moment. Recently, several studies have demonstrated the role of epigenetic mechanisms for control of coordinated expression of genes. In this review, we discuss the contribution of epigenomics to the understanding of gene regulation in Toxoplasma gondii. Studying the distribution of modified histones on the genome links chromatin modifications to gene expression or gene repression. In particular, coincident trimethylated lysine 4 on histone H3 (H3K4me3), acetylated lysine 9 on histone H3 (H3K9ac), and acetylated histone H4 (H4ac) mark promoters of actively transcribed genes. However, the presence of these modified histones at some non‐expressed genes and other histone modifications at only a subset of active promoters implies the presence of other layers of regulation of chromatin structure in T. gondii. Epigenomics analysis provides a powerful tool to characterize the activation state of genomic loci of T. gondii and possibly of other Apicomplexa including Plasmodium or Cryptosporidium. Further, integration of epigenetic data with expression data and other genome‐wide datasets facilitates refinement of genome annotation based upon experimental data.     

A comprehensive genome‐scale reconstruction of Escherichia coli metabolism—2011

The initial genome‐scale reconstruction of the metabolic network of Escherichia coli K‐12 MG1655 was assembled in 2000. It has been updated and periodically released since then based on new and curated genomic and biochemical knowledge. An update has now been built, named iJO1366, which accounts for 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. iJO1366 was (1) updated in part using a new experimental screen of 1075 gene knockout strains, illuminating cases where alternative pathways and isozymes are yet to be discovered, (2) compared with its predecessor and to experimental data sets to confirm that it continues to make accurate phenotypic predictions of growth on different substrates and for gene knockout strains, and (3) mapped to the genomes of all available sequenced E. coli strains, including pathogens, leading to the identification of hundreds of unannotated genes in these organisms. Like its predecessors, the iJO1366 reconstruction is expected to be widely deployed for studying the systems biology of E. coli and for metabolic engineering applications.     

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties

Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H-chain and L-chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long-lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.     

Environmentally friendly approaches to genetic engineering

Summary Several environmental problems related to plant genetic engineering may prohibit advancement of this technology and prevent realization of its full potential. One such common concern is the demonstrated escape of foreign genes through pollen dispersal from transgenic crop plants to their weedy relatives, creating super weeds or causing gene pollution among other crops or toxicity of transgenic pollen to nontarget insects. The high rates of gene flow from crops to wild relatives (as high as 38% in sunflower and 50% in strawberries) are certainly a serious concern. Maternal inheritance of the herbicide resistance gene via chloroplast genetic engineering has been shown to be a practical solution to these problems. Another common concern is the suboptimal production of Bacillus thuringiensis (Bt) insecticidal protein or reliance on a single (or similar) B.t. protein in commercial transgenic crops, resulting in B.t. resistance among target pests. Clearly, different insecticidal proteins should be produced in lethal quantities to decrease the development of resistance. Such hyperexpression of a novel B.t. protein in chloroplasts has resulted in 100% mortality of insects that are up to 40 000-fold resistant to other B.t. proteins. Yet another concern is the presence of antibiotic resistance genes in transgenic plants that could inactivate oral doses of the antibiotic or be transferred to pathogenic microbes in the GI tract or in soil, rendering them resistant to treatment with such antibiotics. Cotransformation and elimination of antibiotic resistant genes from transgenic plants using transposable elements via breeding are promising new approaches. Genetic engineering efforts have also addressed yet another concern, i.e., the accumulation and persistence of plastics in our environment by production of biodegradable plastics. Recent approaches and accomplishments in addressing these environmental concerns via chloroplast genetic engineering are discussed in this review.     

The Genomic Structure of an Insertional Mutation in the Dystonia Musculorum Locus

We have previously identified a line of transgenic mice, Tg4, in which an hsp68-lacZ hybrid gene has inserted into the dystonia musculorum (dt) locus on chromosome 1. We have confirmed the localization of the Tg4 integration site to the proximal region of mouse chromosome 1 by interspecific backcross analysis. One end of the integration complex has been cloned and we have used single-copy probes from the flanking region to screen a mouse genomic library. Several overlapping lambda phage clones have been isolated and arranged into a contig spanning 75 kb of genomic DNA. Probes from the genomic contig have enabled us to characterize the wildtype and Tg4 loci. We report that the integration of the transgene was accompanied by a deletion of 45 kb of host genomic sequences with no other detectable rearrangement in the Tg4 genome.     

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.     

Genomically and biochemically accurate metabolic reconstruction of Methanosarcina barkeri Fusaro,iMG746

Methanosarcina barkeri is an Archaeon that produces methane anaerobically as the primary byproduct of its metabolism. M. barkeri can utilize several substrates for ATP and biomass production including methanol, acetate, methyl amines, and a combination of hydrogen and carbon dioxide. In 2006, a metabolic reconstruction of M. barkeri, iAF692, was generated based on a draft genome annotation. The iAF692 reconstruction enabled the first genome-Scale simulations for Archaea. Since the publication of the first metabolic reconstruction of M. barkeri, additional genomic, biochemical, and phenotypic data have clarified several metabolic pathways. We have used this newly available data to improve the M. barkeri metabolic reconstruction. Modeling simulations using the updated model, iMG746, have led to increased accuracy in predicting gene knockout phenotypes and simulations of batch growth behavior. We used the model to examine knockout lethality data and make predictions about metabolic regulation under different growth conditions. Thus, the updated metabolic reconstruction of M. barkeri metabolism is a useful tool for predicting cellular behavior, studying the methanogenic lifestyle, guiding experimental studies, and making predictions relevant to metabolic engineering applications.     

Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.     

Regulation of INSIG2 by microRNA-96

Mature forms of the microRNAs miR-96, -182, and -183 originate from a single genomic locus and have been shown to be elevated approximately 50-fold in the livers of sterol regulatory element-binding protein-1a and -2 (SREBP-1a and -2) transgenic mice. Our study attempted to identify the possible targets of these microRNAs using miRNA target prediction software. This revealed putative sites in insulin-induced genes (INSIGs). The 3′ untranslated region (UTR) of insulin-induced gene 1 (INSIG1) contained sites corresponding to miR-182, and -183, while the 3′ UTR of INSIG2 featured an miR-96 site. Among these putative sites, only miR-96 demonstrated an inhibitory effect that was specific to the 3′ UTR of INSIG2. As INSIG proteins are the main components of SREBP cleavage complexes that act to release active SREBPs, we assessed the effects of miR-96 on INSIG and SREBP levels and activities. We found that miR-96 reduced the levels of INSIG2 in INSIG1 knockout human fibroblasts, resulting in an increase in SREBP-1 and -2 nuclear forms and a subsequent increase in the abundance of the mRNA of their target genes. These results suggest that miR-96, an miRNA induced by SREBP-2 activation, regulates downstream targets of SREBPs and may increase the abundance of active SREBP.