Application of Transposon Systems in The Transgenesis of Bovine Somatic And Germ Cells

2021 ◽  
Author(s):  
Dong-Hyeok Kwon ◽  
Gyeong-Min Gim ◽  
Kyeong-Hyeon Eom ◽  
Ji-Hyun Lee ◽  
Goo Jang
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Effective Means ◽  
Somatic Cells ◽  
Sleeping Beauty ◽  
Gene Engineering ◽  
Fluorescent Microscope ◽  
Embryo Cells ◽  
Embryo Stage ◽  
Green Fluorescent

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.

A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

2005 ◽  
Vol 342 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Dineshkumar H. Dandekar ◽  
Manish Kumar ◽  
Jayashree S. Ladha ◽  
Krishna N. Ganesh ◽  
Debashis Mitra
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Method ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Green Fluorescent

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

scholarly journals Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

2018 ◽  
Vol 2 (4) ◽  
pp. 671-678
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Foreign Gene ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

scholarly journals Migration of primordial germ cells during late embryogenesis of pikeperch Sander lucioperca relative to blastomere transplantation

2017 ◽  
Vol 62 (No. 3) ◽  
pp. 121-129 ◽  
Author(s):  
H. Güralp ◽  
K. Pocherniaieva ◽  
M. Blecha ◽  
T. Policar ◽  
M. Pšenička ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Somatic Cells ◽  
Time Lapse ◽  
Sander Lucioperca ◽  
Initial Appearance ◽  
Green Fluorescent ◽  
Time Lapse Imaging

Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3’untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3’UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

scholarly journals Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1695
Author(s):  
Alexey Kuzmich ◽  
Olga Rakitina ◽  
Dmitry Didych ◽  
Victor Potapov ◽  
Marina Zinovyeva ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Cancer Associated Fibroblasts ◽  
Histone H2a ◽  
Green Fluorescent

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation

2019 ◽  
Vol 19 (4) ◽  
pp. 248-254
Author(s):  
Marianne Lehmann ◽  
Martina Canatelli-Mallat ◽  
Priscila Chiavellini ◽  
Gloria M. Cónsole ◽  
Maria D. Gallardo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Cell Types ◽  
Cell Reprogramming ◽  
Green Fluorescent ◽  
Original Cell

Background: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. Objective: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. Methods: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). Results: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. Conclusion: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

Protoplast-Based Transient Expression Combined with Plant Cultivation Systems as a Valuable Tool for Floral Thermogenesis Studies in Aroids

2021 ◽  
Author(s):  
Haruhiko Maekawa ◽  
Miyabi Otsubo ◽  
Koichiro Mizoguchi ◽  
Daiki Koyamatsu ◽  
Takehito Inaba ◽  
...  
Keyword(s):  
Transient Expression ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Protein Localization ◽  
Gradient Centrifugation ◽  
Large Family ◽  
Shoot Tip ◽  
Skunk Cabbage ◽  
Green Fluorescent ◽  
Leaf Protoplasts

Abstract Floral thermogenesis in plants plays a significant role in their reproductive function. Thermogenic aroids constitute a large family in highly thermogenic angiosperms, many of which possess intense heat-producing abilities. Several genes have been proposed to be involved in floral thermogenesis of aroids, but the biological tools to identify the functions of those genes at cellular and molecular levels are lacking. Among the many thermogenic aroids, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense, durable heat and small aboveground parts compared with other thermogenic aroids. In this study, leaf protoplasts were isolated from potted and shoot tip-cultured skunk cabbage plants and used to develop transient assay systems. The isolation protocol included an additional, sucrose gradient centrifugation step, which yielded high-purity protoplasts from both types of plants. The isolation and transfection efficiency of the protoplasts exceeded 1.0 × 105/g fresh weight and 50%, respectively, in both potted and shoot tip-cultured plants. Using this protoplast-based transient expression (PTE) system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). In skunk cabbage leaf protoplasts, these three GFP-fused proteins were localized in MitoTracker-stained mitochondria. However, the green fluorescent particles in protoplasts expressing SrUCPA-GFP were enlarged compared with those in protoplasts expressing SrAOX-GFP and SrNDA1-GFP. Our PTE system is a powerful tool for functional gene analysis not only in thermogenic aroids but also in non-thermogenic aroids.

Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery

Nanomedicine ◽  
2021 ◽  
Author(s):  
Akbar Hasanzadeh ◽  
Fatemeh Radmanesh ◽  
Elaheh Sadat Hosseini ◽  
Iman Hashemzadeh ◽  
Jafar Kiani ◽  
...  
Keyword(s):  
Carbon Dots ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
High Serum ◽  
Spectroscopic Techniques ◽  
Delivery Vector ◽  
One Step ◽  
Diphenyl Tetrazolium Bromide ◽  
Green Fluorescent

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

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