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.
Over the past few decades, gene therapy has gained immense importance in medical research as a promising treatment strategy for diseases such as cancer, AIDS, Alzheimer’s disease, and many genetic ...disorders. When a gene needs to be delivered to a target cell inside the human body, it has to pass a large number of barriers through the extracellular and intracellular environment. This is why the delivery of naked genes and nucleic acids is highly unfavorable, and gene delivery requires suitable vectors that can carry the gene cargo to the target site and protect it from biological degradation. To date, medical research has come up with two types of gene delivery vectors, which are viral and nonviral vectors. The ability of viruses to protect transgenes from biological degradation and their capability to efficiently cross cellular barriers have allowed gene therapy research to develop new approaches utilizing viruses and their different genomes as vectors for gene delivery. Although viral vectors are very efficient, science has also come up with numerous nonviral systems based on cationic lipids, cationic polymers, and inorganic particles that provide sustainable gene expression without triggering unwanted inflammatory and immune reactions, and that are considered nontoxic. In this review, we discuss in detail the latest data available on all viral and nonviral vectors used in gene delivery. The mechanisms of viral and nonviral vector-based gene delivery are presented, and the advantages and disadvantages of all types of vectors are also given.
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.
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.
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.
Muscular dystrophy encompasses a large number of heterogeneous genetic disorders characterized by progressive and devastating muscle wasting. Cell-based replacement strategies aimed at promoting ...skeletal muscle regeneration represent a candidate therapeutic approach to treat muscular dystrophies. Due to the difficulties of obtaining large numbers of stem cells from a muscle biopsy as well as expanding these in vitro, pluripotent stem cells (PSCs) represent an attractive cell source for the generation of myogenic progenitors, given that PSCs can repeatedly produce large amounts of lineage-specific tissue, representing an unlimited source of cells for therapy. In this review, we focus on the progress to date on different methods for the generation of human PSC-derived myogenic progenitor cells, their regenerative capabilities upon transplantation, their potential for allogeneic and autologous transplantation, as well as the specific challenges to be considered for future therapeutic applications.
Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene ...expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.
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