Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as ...the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.
Summary
Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future ...improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.
Significance Statement
A highly efficient, inexpensive and easy to use system for assembling large T‐DNAs (transfer‐DNAs) within the Agrobacterium virulence plasmid was developed and shown to frequently generate high quality transgenic plants.
Abstract
Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that ...paramutation occurs at the P1-rr (red pericarp and red cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr′ state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr′. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr′ epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr′ sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation.
Paramutation is an interaction between alleles resulting in heritable silencing of gene expression. First discovered in maize by R. A. Brink in the 1950's, paramutation occurs in both plants and animals; however, understanding what features specify paramutagenic interactions between alleles and/or transgenes remains limited. Here, Sidorenko et al. used transgenic analyses of the maize pericarp color 1 (p1) gene to show that a unique ~600 bp DNA sequence arranged as direct repeats overlapping with a transcribed enhancer mediates all aspects of paramutation.
Anthocyanins are naturally occurring polyphenolic pigments that give food varied colors. Because of their high antioxidant activities, the consumption of anthocyanins has been associated with the ...benefit of preventing various chronic diseases. However, due to natural evolution or human selection, anthocyanins are found only in certain species. Additionally, the insufficient levels of anthocyanins in the most common foods also limit the optimal benefits. To solve this problem, considerable work has been done on germplasm improvement of common species using novel gene editing or transgenic techniques. This review summarized the recent advances in the molecular mechanism of anthocyanin biosynthesis and focused on the progress in using the CRISPR/Cas gene editing or multigene overexpression methods to improve plant food anthocyanins content. In response to the concerns of genome modified food, the future trends in developing anthocyanin-enriched plant food by using novel transgene or marker-free genome modified technologies are discussed. We hope to provide new insights and ideas for better using natural products like anthocyanins to promote human health.
Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all ...disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors. DNA changes in the rAAV gene cassette affect potency, the expression pattern (cell-specificity), and the production yield. Here, we present a library of rAAV vectors and elements that provide a workflow to design novel vectors. We first performed a meta-analysis on recombinant rAAV elements in clinical trials (2007-2020) for ocular gene therapies. We analyzed 33 unique rAAV gene cassettes used in 57 ocular clinical trials. The rAAV gene therapy vectors used six unique capsid variants, 16 different promoters, and six unique polyadenylation sequences. Further, we compiled a list of promoters, enhancers, and other sequences used in current rAAV gene cassettes in preclinical studies. Then, we give an update on pro-viral plasmid backbones used to produce the gene therapy vectors, inverted terminal repeats, production yield, and rAAV safety considerations. Finally, we assess rAAV transgene and bioactivity assays applied to cells or organoids in vitro, explants ex vivo, and clinical studies.
To facilitate model-informed drug development (MIDD) of adeno-associated virus (AAV) therapy, here we have developed a physiologically based pharmacokinetic (PBPK) model for AAVs following ...preclinical investigation in mice. After 2E11 Vg/mouse dose of AAV8 and AAV9 encoding a monoclonal antibody (mAb) gene, whole-body disposition of both the vector and the transgene mAb was evaluated over 3 weeks. At steady-state, the following tissue-to-blood (T/B) concentration ratios were found for AAV8/9: ∼50 for liver; ∼10 for heart and muscle; ∼2 for brain, lung, kidney, adipose, and spleen; ≤1 for bone, skin, and pancreas. T/B values for mAb were compared with the antibody biodistribution coefficients, and five different clusters of organs were identified based on their transgene expression profile. All the biodistribution data were used to develop a novel AAV PBPK model that incorporates: (i) whole-body distribution of the vector; (ii) binding, internalization, and intracellular processing of the vector; (iii) transgene expression and secretion; and (iv) whole-body disposition of the secreted transgene product. The model was able to capture systemic and tissue PK of the vector and the transgene-produced mAb reasonably well. Pathway analysis of the PBPK model suggested that liver, muscle, and heart are the main contributors for the secreted transgene mAb. Unprecedented PK data and the novel PBPK model developed here provide the foundation for quantitative systems pharmacology (QSP) investigations of AAV-mediated gene therapies. The PBPK model can also serve as a quantitative tool for preclinical study design and preclinical-to-clinical translation of AAV-based gene therapies.
It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported ...endogenous reference gene
was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed
gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of
and
with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the
and
genes varied from 1.18 to 2.94%. The copy numbers of the transgene
using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T
and T
progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new
gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.
Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of ...global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T
generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T
lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80-100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25-80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.
Sugarcane (Saccharum spp. hybrids) accounts for 80% of the table sugar produced worldwide and is also a prime feedstock for biofuel production. However, very few studies are available for directly ...comparing Agrobacterium tumefaciens-mediated transfer of T-DNA (AMT) and biolistic transfer of minimal expression cassettes (BLT MC) regarding transgene complexity and expression stability. In this study, the transformation efficiency, transgene integration pattern, expression level, and expression stability were compared in the commercially important sugarcane cultivar CP88-1762. A total of 312 transgenic lines derived from AMT and 250 lines derived from BLT MC were identified by PCR from genomic DNA using nptII-specific primers. Lines were analyzed with both qPCR (TaqMan®) and NPTII ELISA to determine the nptII transgene copy number and expression level. The results of Southern blot analysis on selected lines were highly correlated to the qPCR results. There were no significant differences between the two transformation systems for transformation efficiency, frequency of single copy integration, or level and stability of transgene expression when carried out with the same expression cassette, tissue culture, and selection procedure in 12 independent experiments. These findings suggested that both BLT MC and AMT provide suitable platforms for generation of elite sugarcane events.
•Immune responses may be detrimental to both the safety and efficacy of gene therapy.•Design and manufacturing can be tuned to reduce vector immunogenicity.•Active transgene-specific immune tolerance ...is desirable in gene therapy.•Novel targeted immune-modulatory strategies can be explored to improve gene therapy.
Lentiviral vectors (LV) are widely used vehicles for gene transfer and therapy in pre-clinical animal models and clinical trials with promising safety and efficacy results. However, host immune responses against vector- and/or transgene-derived antigens remain a major obstacle to the success and broad applicability of gene therapy. Here we review the innate and adaptive immunological barriers to successful gene therapy, both in the context of ex vivo and in vivo LV gene therapy, mostly concerning systemic LV delivery and discuss possible means to overcome them, including vector design and production and immune modulatory strategies.
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