gene engineering
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2021 ◽  
Author(s):  
Dong-Hyeok Kwon ◽  
Gyeong-Min Gim ◽  
Kyeong-Hyeon Eom ◽  
Ji-Hyun Lee ◽  
Goo Jang

Abstract Background: Several DNA transposons, PiggyBac (PB), Sleeping beauty (SB) and Tol2 have been applied as effective means for transgenesis in many species. Cattle are not typical experimental animals, and relatively little verification has been studied in this species. Thus, the goal of this study was the applicability of three transposon systems in somatic and embryo cells in cattle, while also determining which of the three systems is appropriate for each type of cell. To conduct the experiment, green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, GFP-positive cells or blastocysts were observed through a fluorescent microscope and transfection efficiency was calculated by FACS.Results: In the bovine somatic cells experiment, the PB (63.97 ± 11.56) showed higher efficiency as compared to the other two systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Unlike the results of the somatic cells, Tol2 (75.00%) and SB (70.00%) in the embryo were more efficient as compared to PB (42.86 %).Conclusions: These results demonstrate that all three transposon systems can be used in bovine somatic cells and embryos as a gene engineering experimental method and which type of transposon system is appropriate to apply depending on the cell type.


Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6000
Author(s):  
Daniel Fowler ◽  
Callum Nattress ◽  
Alba Southern Navarrete ◽  
Marta Barisa ◽  
Jonathan Fisher

Although chimeric antigen receptor (CAR) T cells have shown impressive clinical success against haematological malignancies such as B cell lymphoma and acute lymphoblastic leukaemia, their efficacy against non-haematological solid malignancies has been largely disappointing. Solid tumours pose many additional challenges for CAR T cells that have severely blunted their potency, including homing to the sites of disease, survival and persistence within the adverse conditions of the tumour microenvironment, and above all, the highly immunosuppressive nature of the tumour milieu. Gene engineering approaches for generating immune cells capable of overcoming these hurdles remain an unmet therapeutic need and ongoing area of research. Recent advances have involved gene constructs for membrane-bound and/or secretable proteins that provide added effector cell function over and above the benefits of classical CAR-mediated cytotoxicity, rendering immune cells not only as direct cytotoxic effectors against tumours, but also as vessels for payload delivery capable of both modulating the tumour microenvironment and orchestrating innate and adaptive anti-tumour immunity. We discuss here the novel concept of engineered immune cells as vessels for payload delivery into the tumour microenvironment, how these cells are better adapted to overcome the challenges faced in a solid tumour, and importantly, the novel gene engineering approaches required to deliver these more complex polycistronic gene constructs.


Author(s):  
Wilfried A Kues ◽  
Dharmendra Kumar ◽  
Naresh L Selokar ◽  
Thirumala Rao Talluri

: Precise and site specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulating of any gene in various types of cells with target specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, make alterations and cure diseases permanently that are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also shown to have a significant improvement in clinical trials. However, their widespread use in research revealed potential safety issues, which need to be addressed before implementing such techniques in clinical purposes. Significant and valiant attempts are being made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.


2021 ◽  
Vol 22 (19) ◽  
pp. 10239
Author(s):  
Huai Yang ◽  
Shengfu Zhong ◽  
Chen Chen ◽  
Hao Yang ◽  
Wei Chen ◽  
...  

Wheat powdery mildew, caused by the obligate parasite Blumeria graminis f. sp. tritici, severely reduces wheat yields. Identifying durable and effective genes against wheat powdery mildew and further transferring them into wheat cultivars is important for finally controlling this disease in wheat production. Pm40 has been widely used in wheat breeding programs in Southwest China due to the spectrum and potentially durable resistance to powdery mildew. In the present study, a resistance test demonstrated that Pm40 is still effective against the Bgt race E20. We identified and cloned the TraesCS7B01G164000 with a total length of 4883 bp, including three exons and two introns, and encoded a protein carrying the CC-NBS-NBS-LRR domain in the Pm40-linked region flanked by two EST markers, BF478514 and BF291338, by integrating analysis of gene annotation in wheat reference genome and both sequence and expression difference in available transcriptome data. Two missense mutations were detected at positions 68 and 83 in the CC domain. The results of both cosegregation linkage analysis and qRT-PCR also suggested that TraesCS7B01G164000 was a potential candidate gene of Pm40. This study allowed us to move toward the final successfully clone and apply Pm40 in wheat resistance improvement by gene engineering.


Author(s):  
Qin Jiang ◽  
Keli Huang ◽  
Fang Lu ◽  
Shaoping Deng ◽  
Zhenglin Yang ◽  
...  

AbstractMesenchymal stem cell (MSC) transplantation is regarded as a promising candidate for the treatment of ischaemic heart disease. The major hurdles for successful clinical translation of MSC therapy are poor survival, retention, and engraftment in the infarcted heart. Stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4) constitutes one of the most efficient chemokine/chemokine receptor pairs regarding cell homing. In this review, we mainly focused on previous studies on how to regulate the SDF-1/CXCR4 interaction through various priming strategies to maximize the efficacy of mesenchymal stem cell transplantation on ischaemic hearts or to facilitate the required effects. The strengthened measures for enhancing the therapeutic efficacy of the SDF-1/CXCR4 interaction for mesenchymal stem cell transplantation included the combination of chemokines and cytokines, hormones and drugs, biomaterials, gene engineering, and hypoxia. The priming strategies on recipients for stem cell transplantation included ischaemic conditioning and device techniques.


2021 ◽  
Vol 4 (3) ◽  
Author(s):  
Mahshid Mehdizadeh ◽  
Samira Karami ◽  
Haniyeh Ghaffari Nazari ◽  
Ghazaleh Sankanian ◽  
Mohsen Hamidpour ◽  
...  
Keyword(s):  
T Cells ◽  

2021 ◽  
Vol 11 (2) ◽  
pp. 123-139
Author(s):  
Mikhail Yur. Cherednichenko ◽  
Oksana B. Polivanova ◽  
Daria An. Khlebnikova ◽  
Anastasiya Vl. Sosina ◽  
Mariia M. Belova ◽  
...  

Gene engineering is a convenient way of solving basic and applied tasks related to the system of secondary metabolism of medicinal and aromatic plants, such as a large and economically important plant family Lamiaceae Martinov (Labiatae Juss.) that includes such major species, as Agastache, Dracocephalum, Lavandula, Mentha, Ocimum, Salvia, Satureja, Thymus, etc. The basic value of this method consists in studying the genetic mechanism of secondary metabolism, which includes key enzyme genes, transcription factors, etc. Applied research consists in producing valuable secondary metabolites with biological activity, including active pharmaceutical ingredients in both, the familyʹs plants proper and the heterologous systems using relevant plant genes. This overview considers a set of diverse gene engineering studies.


2021 ◽  
Author(s):  
Dorottya A. Simon ◽  
András Tálas ◽  
Péter I. Kulcsár ◽  
Ervin Welker

ABSTRACTPrime editing is a recently developed gene engineering tool that allows the introduction of short insertions, deletions or substitutions into the genome. However, the efficiency of prime editing, generally reaching around 10-30% editing, has not resembled its versatility. Here, Prime Editor Activity Reporter (PEAR), a sensitive fluorescent tool is introduced for the identification of single cells with prime editing activity. Possessing no background fluorescence, PEAR specifically reports on prime editing events in individual cells. By design, it ensures unrestricted flexibility for sequence variations in the full length of the target sequence. The application of PEAR as an enrichment marker of prime editing can increase the edited population by up to 70% and alleviate the burden of the otherwise time and labour consuming process of cloning of the correctly edited cells, therefore considerably improving the applicability of prime editing in fundamental research and biotechnological uses.


2021 ◽  
Vol 12 ◽  
Author(s):  
Tingting Feng ◽  
Sebastian Leptihn ◽  
Ke Dong ◽  
Belinda Loh ◽  
Yan Zhang ◽  
...  

Phage therapy represents a possible treatment option to cure infections caused by multidrug-resistant bacteria, including methicillin and vancomycin-resistant Staphylococcus aureus, to which most antibiotics have become ineffective. In the present study, we report the isolation and complete characterization of a novel phage named JD219 exhibiting a broad host range able to infect 61 of 138 clinical strains of S. aureus tested, which included MRSA strains as well. The phage JD419 exhibits a unique morphology with an elongated capsid and a flexible tail. To evaluate the potential of JD419 to be used as a therapeutic phage, we tested the ability of the phage particles to remain infectious after treatment exceeding physiological pH or temperature. The activity was retained at pH values of 6.0–8.0 and below 50°C. As phages can contain virulence genes, JD419’s complete genome was sequenced. The 45509 bp genome is predicted to contain 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Genome analysis indicates that JD419 is a temperate phage, despite observing rapid replication and lysis of host strains. Following the recent advances in synthetic biology, JD419 can be modified by gene engineering to remove prophage-related genes, preventing potential lysogeny, in order to be deployed as a therapeutic phage.


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