Prime editing enables search-and-replace genome editing but is limited by low editing efficiency. We present a high-throughput approach, the Peptide Self-Editing sequencing assay (PepSEq), to measure ...how fusion of 12,000 85-amino acid peptides influences prime editing efficiency. We show that peptide fusion can enhance prime editing, prime-enhancing peptides combine productively, and a top dual peptide-prime editor increases prime editing significantly in multiple cell lines across dozens of target sites. Top prime-enhancing peptides function by increasing translation efficiency and serve as broadly useful tools to improve prime editing efficiency.
In embryonic development, the pancreas and liver share developmental history up to the stage of bud formation. Therefore, we postulated that direct reprogramming of liver to pancreatic cells can ...occur when suitable transcription factors are overexpressed. Using a polycistronic vector we misexpress Pdx1, Ngn3, and MafA in the livers of NOD-SCID mice rendered diabetic by treatment with streptozotocin (STZ). The diabetes is relieved long term. Many ectopic duct-like structures appear that express a variety of β-cell markers, including dense core granules visible by electron microscopy (EM). Use of a vector also expressing GFP shows that the ducts persist long after the viral gene expression has ceased, indicating that this is a true irreversible cell reprogramming event. We have recovered the insulin⁺ cells by cell sorting and shown that they display glucose-sensitive insulin secretion. The early formed insulin⁺ cells can be seen to coexpress SOX9 and are also labeled in mice lineage labeled for Sox9 expression. SOX9⁺ cells are normally found associated with small bile ducts in the periportal region, indicating that the duct-like structures arise from this source. This work confirms that developmentally related cells can be reprogrammed by suitable transcription factors and also suggests a unique therapy for diabetes.
Pfs48/45 is a leading antigen candidate for a transmission blocking (TB) vaccine. However, efforts to produce affordable, safe and correctly folded full-length Pfs48/45 using different protein ...expression systems have not produced an antigen with satisfactory TB activity. Pfs48/45 has 16 cysteines involved in disulfide bond formation, and the correct formation is critical for proper folding and induction of TB antibodies. Moreover, Pfs48⁄45 is not a glycoprotein in the native hosts, but contains potential glycosylation sites, which are aberrantly glycosylated during expression in eukaryotic systems. Here, we demonstrate for the first time that full length, Endo H in vivo enzymatic deglycosylated Pfs48/45 antigen is produced at a high level in plants and is structurally stable at elevated temperatures. Sera from mice immunized with this antigen showed strong inhibition in SMFA. Thus, Endo H in vivo enzymatic deglycosylated Pfs48/45 is a promising candidate for the development of an affordable TB vaccine, which may have the potential to save millions.
Pdx1 (pancreatic and duodenal homeobox 1), Ngn3 (neurogenin 3) and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A) have been reported to bring about the transdifferentiation ...of pancreatic exocrine cells to beta (β) cells in vivo. We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM (adenoviral Pdx1, Ngn3, MafA construct). When introduced into AR42j-B13 cells, Ad-PNM caused a rapid change to a flattened morphology and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are up-regulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 (insulin 1) and Ins2 (insulin 2) gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID (non-obese diabetic severe combined immunodeficiency) mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.
The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and ...SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Anatolia is one of the important production areas of garlic. Garlic production is conducted by using the head or the cloves inside the heads, which are actually the consumed part of the garlic, as ...propagation material. However, due to the use of cloves, which are the most valuable part of the market, as reproduction material, the profit is reduced by about 10%. The study aims to provide an alternative propagation material to reduce the losses resulting from this practice in local Kahramanmaraş garlic production. For this purpose, generating plantlets directly from stem discs of the garlic cloves in vitro and the effects of different growth regulators have been studied. For this, the plant growth regulators added to MS media as BAP (1.0, 1.5 and 2.0 mg l-1), GA3 (0.5, 1.0 and 1.5 mg l-1), 2-IP (0.75, 1.00 and 1.25 mg l-1); kinetin (1.0, 2.0 and 3.0 mg l-1) and TDZ (0.75, 1.00 and 1.25 mg l-1) were tested. In the study number of explants, number of infected explants, number of healthy explants, number of developed explants, healthy explant rate, developed explant rate, number of callused explants, callus growth rate, number of proliferated explants, proliferation rate, proliferation number, number of rooted explants, rooting rate and number of roots were investigated. However, shoot ratios, shoot numbers, and callus formation were the main focus. The highest rates of proliferation were found in 2-IP (53.8%, 45.5%, and 40.0% at 1.00, 0.75, and 1.25 mg l-1 dosages, respectively) and Kinetin (35.3% at 2.00 mg l-1). The maximum shoot number was reached with 2-IP at the dose of 1.00 mg l-1 as 1.9 shoot/explants. Kinetin at 3.00 mg l-1 and 2IP at 1.25 mg l-1 were the other successful applications with 1.8 shoots. This study indicated promising results to obtain plantlets directly from the clove’s stem discs and including them into seedling production for the mass production of garlic.
Anadolu sarımsağın önemli üretim alanlarından birisidir. Sarımsak üretimi, sarımsağın tüketilen kısmı olan baş veya başların içindeki dişlerin üretim materyali olarak kullanılmasıyla gerçekleştirilmektedir. Ancak, pazar bakımından en değerli kısmı olan dişlerin üreme materyali olarak kullanılması nedeniyle kazanç yaklaşık %10 oranında azalmaktadır. Araştırmanın amacı Kahramanmaraş yöresel sarımsak üretiminde, dişlerle yapılan üretim sonucu ortaya çıkan kayıpları azaltmak için alternatif çoğaltma materyali sağlanmasıdır. Bu amaçla sarımsak dişlerinin gövde disklerinden in vitro’da doğrudan bitkicik elde edilmesine ve farklı bitki büyüme düzenleyicilerin etkilerine çalışılmıştır. Bunun için MS besi ortamına eklenen bitki büyüme düzenleyicilerden BAP (1.0, 1.5 ve 2.0 mg l-1), GA3 (0.5, 1.0 ve 1.5 mg l-1), 2-IP (0.75, 1.00 ve 1.25 mg l-1), Kinetin (1.0, 2.0 ve 3.0 mg l-1) ve TDZ (0.75, 1.00 ve 1.25 mg l-1) test edilmiştir. Çalışmada eksplant sayısı, enfekte eksplant sayısı, sağlıklı eksplant sayısı, gelişmiş eksplant sayısı, sağlıklı eksplant oranı, gelişmiş eksplant oranı, nasırlı eksplant sayısı, kallus büyüme hızı, çoğalan eksplant sayısı, çoğalma hızı, çoğalma sayısı, köklenen eksplant sayısı, köklenme oranı ve kök sayısı incelenmiştir. Ancak daha çok sürgün oranları, sürgün sayıları ve kallus oluşumu üzerinde durulmuştur. En yüksek kardeşlenme oranları 2-IP (1.00, 0.75 ve 1.25 mg l-1’de sırasıyla % 53.8, % 45.5 ve % 40.0) ve Kinetin (2.00 mg l-1’de % 35.3)’de sağlanmıştır. En fazla kardeş sayısına 1.9 adet kardeş/eksplant ile 1.00 mg l-1 2-IP dozunda ulaşılmıştır. Kinetin’nin 3.00 mg l-1 ve 2-IP’nin 1.25 mg l-1 dozları 1.8 sürgün ile başarılı sonuçlar alınan diğer uygulamalar olmuştur. Bu çalışma, sarımsağın yoğun üretiminde, dişlerin gövde disklerinden doğrudan bitkicik elde edilmesinde ve bunların üretime fide olarak dahil edilmesinde umut verici sonuçlara işaret etmektedir.
Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely ...through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.
A common pathological hallmark of neurodegenerative disorders is neuronal cell death, accompanied by neuroinflammation and oxidative stress. The vasoactive intestinal peptide (VIP) is a pleiotropic ...peptide that combines neuroprotective and immunomodulatory actions. The gene therapy field shows long‐term promise for treating a wide range of neurodegenerative diseases (ND). In this study, we aimed to investigate the in vitro efficacy of transduction of microglia using lentiviral gene therapy vectors encoding VIP (LentiVIP). Additionally, we tested the protective effects of the secretome derived from LentiVIP‐infected “immortalized human” microglia HMC3 cells, and cells treated with Synthetic VIP (SynVIP), against toxin‐induced neurodegeneration. First, LentiVIP, which stably expresses VIP, was generated and purified. VIP secretion in microglial conditioned media (MG CM) for LentiVIP‐infected HMC3 microglia cells was confirmed. Microglia cells were activated with lipopolysaccharide, and groups were formed as follows: 1) Control, 2) SynVIP‐treated, or 3) LentiVIP‐transduced. These MG CM were applied on an in vitro neurodegenerative model formed by differentiated (d)‐SH‐SY5Y cells. Then, cell survival analysis and apoptotic nuclear staining, besides measurement of oxidative/inflammatory parameters in CM of cells were performed. Activated MG CM reduced survival rates of both control and toxin‐applied (d)‐SH‐SY5Y cells, whereas LentiVIP‐infected MG CM and SynVIP‐treated ones exhibited better survival rates. These findings were supported by apoptotic nuclear evaluations of (d)‐SH‐SY5Y cells, alongside oxidative/inflammatory parameters in their CM. LentiVIP seems worthy of further studies for the treatment of ND because of the potential of gene therapy to treat diseases effectively with a single injection.
Cultured microglial cells were activated with LPS, and then grouped as: 1) Control, 2) SynVIP‐treated, or 3) LentiVIP‐transduced microglia. Subsequently, conditioned media of microglial groups were applied on differentiated‐SH‐SY5Y cells that were exposed to MPTP (a neurotoxin) or not. Several analysis and tests were performed to investigate the protective effects of SynVIP or LentiVIP against toxicity of MPTP or microglial secretome.
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