DNA Engineering and Hepatitis B Virus Replication

Recombinant DNA technology is a vital method in human hepatitis B virus (HBV), producing reporter viruses or vectors for gene transferring. Researchers have engineered several genes into the HBV genome for different purposes; however, a systematic analysis of recombinant strategy is lacking. Here, using a 500-bp deletion strategy, we scanned the HBV genome and identified two regions, region I (from nt 2,118 to 2,814) and region II (from nt 99 to 1,198), suitable for engineering. Ten exogenous genes, including puromycin N-acetyl transferase gene (Pac), blasticidin S deaminase gene (BSD), Neomycin-resistance gene (Neo), Gaussia luciferase (Gluc), NanoLuc (Nluc), copGFP, mCherry, UnaG, eGFP, and tTA1, were inserted into these two regions and fused into the open reading frames of hepatitis B core protein (HBC) and hepatitis B surface protein (HBS) via T2A peptide. Recombination of 9 of the 10 genes at region 99–1198 and 5 of the 10 genes at region 2118–2814 supported the formation of relaxed circular (RC) DNA. HBV DNA and HBV RNA assays implied that exogenous genes potentially abrogate RC DNA by inducing the formation of adverse secondary structures. This hypothesis was supported because sequence optimization of the UnaG gene based on HBC sequence rescued RC DNA formation. Findings from this study provide an informative basis and a valuable method for further constructing and optimizing recombinant HBV and imply that DNA sequence might be intrinsically a potential source of selective pressure in the evolution of HBV.

Intracellular Low Iron Exerts Anti-BK Polyomavirus Effect by Inhibiting the Protein Synthesis of Exogenous Genes

BKPyV poses a serious threat to the health of immunocompromised patients, and there are currently no curative drugs. Understanding the relationship between the virus and intracellular environment contributes to the discovery of antiviral targets.

Spatial and temporal control of expression with light-gated LOV-LexA

The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has catapulted discovery in biology. While many enhancers drive expression broadly, several genetic tricks have been developed to obtain access to isolated cell types. However, studies of topographically organized neuropiles, such as the optic lobe in fruit flies, have raised the need for a system that can access subsets of cells within a single neuron type, a feat currently dependent on stochastic flip-out methods. To access the same subsets of cells consistently across flies, we developed LOV-LexA, a light-gated expression system based on the bacterial LexA transcription factor and the plant-derived LOV photosensitive domain. Expression of LOV-Lex in larval fat body as well as pupal and adult neurons enables spatial and temporal control of expression of transgenes under LexAop sequences with blue light. The LOV-LexA tool thus provides another layer of intersectional genetics, allowing for light-controlled genetic access to the same subsets of cells within an expression pattern across individual flies.

Feature selection for RNA cleavage efficiency at specific sites using the LASSO regression model in Arabidopsis thaliana

Background RNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana. Results Cleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model. The results indicated that whole RNA features were important for the CSsite value, in addition to features around cleavage sites. Whole RNA features related to the translation process and nucleotide frequency around cleavage sites were major determinants of cleavage efficiency. The results were verified in a model constructed using only sequence features, which showed that the prediction accuracy was similar to that determined using all features including the translation process, suggesting that cleavage efficiency can be predicted using only sequence information. The LASSO regression model was validated in exogenous genes, which showed that the model constructed using only sequence information can predict cleavage efficiency in both endogenous and exogenous genes. Conclusions Feature selection using the LASSO regression model in A. thaliana identified 155 features. Correlation coefficients revealed that whole RNA features are important for determining cleavage efficiency in addition to features around the cleavage sites. The LASSO regression model can predict cleavage efficiency in endogenous and exogenous genes using only sequence information. The model revealed the significance of the effect of multiple determinants on cleavage efficiency, suggesting that sequence features are important for RNA degradation mechanisms in A. thaliana.

Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic Arabidopsis

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

Strategy for generation of replication–competent recombinant rotaviruses expressing multiple foreign genes

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

DNA Methylation Silences Exogenous Gene Expression in Transgenic Birch Progeny

The genetic stability of exogenous genes in the progeny of transgenic trees is extremely important in forest breeding; however, it remains largely unclear. We selected transgenic birch (Betula platyphylla) and its hybrid F1 progeny to investigate the expression stability and silencing mechanism of exogenous genes. We found that the exogenous genes of transgenic birch could be transmitted to their offspring through sexual reproduction. The exogenous genes were segregated during genetic transmission. The hybrid progeny of transgenic birch WT1×TP22 (184) and WT1×TP23 (212) showed higher Bgt expression and greater insect resistance than their parents. However, the hybrid progeny of transgenic birch TP23×TP49 (196) showed much lower Bgt expression, which was only 13.5% of the expression in its parents. To elucidate the mechanism underlying the variation in gene expression between the parents and progeny, we analyzed the methylation rates of Bgt in its promoter and coding regions. The hybrid progeny with normally expressed exogenous genes showed much lower methylation rates (0–29%) than the hybrid progeny with silenced exogenous genes (32.35–45.95%). These results suggest that transgene silencing in the progeny is mainly due to DNA methylation at cytosine residues. We further demonstrated that methylation in the promoter region, rather than in the coding region, leads to gene silencing. We also investigated the relative expression levels of three methyltransferase genes: BpCMT, BpDRM, and BpMET. The transgenic birch line 196 with a silenced Gus gene showed, respectively, 2.54, 9.92, and 4.54 times higher expression levels of BpCMT, BpDRM, and BpMET than its parents. These trends are consistent with and corroborate the high methylation levels of exogenous genes in the transgenic birch line 196. Therefore, our study suggests that DNA methylation in the promoter region leads to silencing of exogenous genes in transgenic progeny of birch.

Identification of the enzymes responsible for m2,2G and acp3U formation on cytosolic tRNA from insects and plants

Posttranscriptional modification of tRNA is critical for efficient protein translation and proper cell growth, and defects in tRNA modifications are often associated with human disease. Although most of the enzymes required for eukaryotic tRNA modifications are known, many of these enzymes have not been identified and characterized in several model multicellular eukaryotes. Here, we present two related approaches to identify the genes required for tRNA modifications in multicellular organisms using primer extension assays with fluorescent oligonucleotides. To demonstrate the utility of these approaches we first use expression of exogenous genes in yeast to experimentally identify two TRM1 orthologs capable of forming N2,N2-dimethylguanosine (m2,2G) on residue 26 of cytosolic tRNA in the model plant Arabidopsis thaliana. We also show that a predicted catalytic aspartate residue is required for function in each of the proteins. We next use RNA interference in cultured Drosophila melanogaster cells to identify the gene required for m2,2G26 formation on cytosolic tRNA. Additionally, using these approaches we experimentally identify D. melanogaster gene CG10050 as the corresponding ortholog of human DTWD2, which encodes the protein required for formation of 3-amino-3-propylcarboxyuridine (acp3U) on residue 20a of cytosolic tRNA. We further show that A. thaliana gene AT2G41750 can form acp3U20b on an A. thaliana tRNA expressed in yeast cells, and that the aspartate and tryptophan residues in the DXTW motif of this protein are required for modification activity. These results demonstrate that these approaches can be used to study tRNA modification enzymes.

Effect of exogenous transcription factors integration sites on safety and pluripotency of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs), generated from somatic cells, not only possess similar characteristics with embryonic stem cells (ESCs), but also present more advantages than ESCs in medical applications. The classical induction method that utilizes the integration of exogenous genes into chromosomes may raise the potential risk of the safety of iPSCs. To investigate the potential correlation between the integration sites of exogenous transcription factors (TFs) and iPSCs’ pluripotency and safety, the integration of exogenous genes in three iPSC lines, which met the golden standard of murine developmental assay (tetraploid complementation), were analyzed. Twenty-two integration sites of exogenous TFs were identified by nested inverse polymerase chain reaction (iPCR) and 39 flanking genes’ functions were analyzed by gene ontology (GO). In the 22 integrated sites, 17 (77.3%) were located in the intergenic regions and the remainder were located in introns far from the transcription start sites. Microarray analysis of the flanking genes in these cells showed that there was no distinct difference in expression levels between the iPSCs, ESCs and mouse embryonic fibroblast (MEF), suggesting that the integration of exogenous TFs has no significant influence on the expression of flanking genes. Gene ontology analysis showed that although most of the flanking genes were housekeeping genes, which were necessary for basic life activity, none of these 39 flanking genes have correlation with tumorigenesis or embryogenesis, suggesting that the integration sites hold low risk of tumorigenesis.