scholarly journals Engraftment of allotransplantated tumour cells in adult rag2 mutant Xenopus tropicalis

2021 ◽  
Author(s):  
Dieter Tulkens ◽  
Dionysia Dimitrakopoulou ◽  
Tom Van Nieuwenhuysen ◽  
Marthe Boelens ◽  
Suzan Demuynck ◽  
...  
Keyword(s):  
Human Cancer ◽  
Genetic Diseases ◽  
Genetically Engineered ◽  
Tumour Cells ◽  
Xenopus Tropicalis ◽  
Tumour Suppressor Genes ◽  
The Novel ◽  
Knock Out ◽  
Early Embryos ◽  
Tumour Formation

Modelling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated. This included the induction of autochthonous tumour formation by injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting multiple tumour suppressor genes. What has been lacking so far is the possibility to propagate the induced cancers via transplantation. In this paper we describe the generation of a rag2-/- knock-out line in Xenopus tropicalis that is deficient in functional T- and B-cells. This line was validated by means of an allografting experiment with a primary tp53-/- donor tumour. In addition, we optimized available protocols for sub-lethal gamma irradiation of X. tropicalis froglets. Irradiated animals also allowed stable, albeit transient, engraftment of transplanted tp53-/- tumour cells. The novel X. tropicalis rag2-/- line and the irradiated wild type froglets will further expand the experimental toolbox in this diploid amphibian, and help to establish it as a versatile and relevant model for exploring human cancer.

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Meng-Yin Li ◽  
Jie Yang ◽  
Ya-Qian Wang ◽  
Xue-Yuan Wu ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Genetic Diseases ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Lesion Detection ◽  
The Novel ◽  
Structural Variations ◽  
Dna Lesion ◽  
Base Lesion

DNA lesion such as metholcytosine(<sup>m</sup>C), 8-OXO-guanine(<sup>O</sup>G), inosine(I) <i>etc</i> could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (<sup>m</sup>C, <sup>O</sup>G, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

Pharmaceutical and Biotechnological Applications of Transgenic Technology

2018 ◽  
Vol 11 (4) ◽  
pp. 4145-4153
Author(s):  
D Samba Reddy ◽  
Tina Reddy
Keyword(s):  
Mammalian Species ◽  
Genetic Diseases ◽  
Transgenic Animals ◽  
Transgenic Animal ◽  
Pharmaceutical Products ◽  
Conditional Knockout ◽  
Knock Out ◽  
Transgenic Lines ◽  
Health And Disease ◽  
Or Genes

A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined.  They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth).  The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine.  Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products.  In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.    

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

2020 ◽  
Vol 14 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Maryam Ahankoub ◽  
Gashtasb Mardani ◽  
Payam Ghasemi-Dehkordi ◽  
Ameneh Mehri-Ghahfarrokhi ◽  
Abbas Doosti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Hazardous Substances ◽  
E Coli ◽  
Spiked Soil ◽  
Engineered Escherichia Coli ◽  
Genetically Manipulated ◽  
Hplc Measurement

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

PLATELET ACTIVATION BY HUMAN CANCER CELLS GROWN “IN VITRO” OR DISSOCIATED FROM TUMOUR TISSUES

1987 ◽  
Author(s):  
G Grignani ◽  
L Pacchiarini ◽  
M Zucchella ◽  
L Dezza ◽  
S C Rizzo
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Cancer ◽  
Iodoacetic Acid ◽  
Tumour Cells ◽  
Human Cancer Cells ◽  
Human Tumour Cells ◽  
Dissociated Cells ◽  
Lymphoblastic Cells

The mechanisms of platelet activation by human tumour cells grown “in vitro” or freshly dissociated from tumour tissues have been investigated.MoCCL human T-lymphoblastic cells cultured “in vitro” induced platelet aggregation through the production of ADP, as evidenced by inhibition of the effect by apyrase. The maximum of ADP production by tumour cells was reached after 1 hour and was 225 p moles/106 cells.On the contrary, platelet aggregation induced by 5637 human bladder carcinoma cells was not inhibited by apyrase, but was abolished by hirudin, indicating the important role of thrombin in this effect.Tumour cells dissociated from 3 breast carcinomas showed a very high platelet aggregating activity, which was not inhibited by hirudin or apyrase, but was abolished by iodoacetic acid, suggesting a role for a cystein-protease in platelet activation.These results confirm that platelets can be activated by tumour cells through different mechanisms; they also suggest that the methods employed to obtain the tumour cells can influence the results, probably because of the different cell populations which are present in the dissociated tumour tissues.Informations obtained with freshly dissociated cells are interesting, because this method has been used seldom so far and because it provides a more physiological approach to the study of the interactions of tumours and platelets.

scholarly journals Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

2016 ◽  
Vol 113 (42) ◽  
pp. E6409-E6417 ◽  
Author(s):  
David G. McFadden ◽  
Katerina Politi ◽  
Arjun Bhutkar ◽  
Frances K. Chen ◽  
Xiaoling Song ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Mouse Models ◽  
Cancer Progression ◽  
Large Scale ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Model Systems ◽  
Driver Mutations ◽  
Genetic Profile ◽  
Genetically Engineered Mouse Models

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

CRISPR–Cas9 gene editing as a tool for developing immunotherapy for cancer

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Gene Editing ◽  
Genetically Engineered ◽  
Cancer Resistance ◽  
Knock Out ◽  
Tumor Tissues ◽  
Engineered T Cells ◽  
Long Time

T cells following genome editing and transformation might be detectable in peripheral blood and tumor tissues for a long time, even more than a year. The types and diversity of T-cells in peripheral blood and tumor tissues changed following transfusion of genetically modified T-cells, and some highly suspected T-cells targeting cancer cells grew, increasing the proportion of such cells. Moreover, after getting genetically engineered T cells, anticancer cytokine secretion increased. T cells changed by gene editing have certain functions, at least from an immunological standpoint. The first clinical research using the CRISPR–Cas9 gene editing method for cancer resistance is more complicated: Using CRISPR–Cas9 gene editing technology to concurrently knock out, amplify, activate and reinfuse three genes in human immune cells. This therapeutic strategy is more demanding, because the changed immune cells have a wider target scope. The data suggest that the efficacy of gene editing in immune cells was 15–45%, and the modified cells could survive long in the peripheral blood and tumor tissues of patients. After three or four months, some T-cells became central T-cells. These encouraging findings pave the way for future experimental cancer research utilizing CRISPR technology.

scholarly journals Mouse models for BRAF-induced cancers

2007 ◽  
Vol 35 (5) ◽  
pp. 1329-1333 ◽  
Author(s):  
C. Pritchard ◽  
L. Carragher ◽  
V. Aldridge ◽  
S. Giblett ◽  
H. Jin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mouse Models ◽  
Human Cancer ◽  
Cre Recombinase ◽  
Genetically Engineered ◽  
Mitogen Activated Protein Kinase ◽  
Braf Mutations ◽  
Single Nucleotide Change

Oncogenic mutations in the BRAF gene are detected in ∼7% of human cancer samples with a particularly high frequency of mutation in malignant melanomas. Over 40 different missense BRAF mutations have been found, but the vast majority (>90%) represent a single nucleotide change resulting in a valine→glutamate mutation at residue 600 (V600EBRAF). In cells cultured in vitro, V600EBRAF is able to stimulate endogenous MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK phosphorylation leading to an increase in cell proliferation, cell survival, transformation, tumorigenicity, invasion and vascular development. Many of these hallmarks of cancer can be reversed by treatment of cells with siRNA (small interfering RNA) to BRAF or by inhibiting MEK, indicating that BRAF and MEK are attractive therapeutic targets in cancer samples with BRAF mutations. In order to fully understand the role of oncogenic BRAF in cancer development in vivo as well as to test the in vivo efficacy of anti-BRAF or anti-MEK therapies, GEMMs (genetically engineered mouse models) have been generated in which expression of oncogenic BRaf is conditionally dependent on the Cre recombinase. The delivery/activation of the Cre recombinase can be regulated in both a temporal and spatial manner and therefore these mouse models can be used to recapitulate the somatic mutation of BRAF that occurs in different tissues in the development of human cancer. The data so far obtained following Cre-mediated activation in haemopoietic tissue and the lung indicate that V600EBRAF mutation can drive tumour initiation and that its primary effect is to induce high levels of cyclin D1-mediated cell proliferation. However, hallmarks of OIS (oncogene-induced senescence) are evident that restrain further development of the tumour.

scholarly journals Expression, purification and crystallization of the novel Xenopus tropicalis ALDH16B1, a homologue of human ALDH16A1

2019 ◽  
Vol 304 ◽  
pp. 168-172 ◽  
Author(s):  
Georgios Pantouris ◽  
Evangelos Dioletis ◽  
Ying Chen ◽  
David C. Thompson ◽  
Vasilis Vasiliou ◽  
...  
Keyword(s):  
Xenopus Tropicalis ◽  
The Novel

scholarly journals Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 628
Author(s):  
Elena McBeath ◽  
Jan Parker-Thornburg ◽  
Yuka Fujii ◽  
Neeraj Aryal ◽  
Chad Smith ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
Genetically Engineered ◽  
Rapid Evaluation ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetically Engineered Mice ◽  
The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

scholarly journals The novel roles of circRNAs in human cancer

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Qingfeng Shang ◽  
Zhi Yang ◽  
Renbing Jia ◽  
Shengfang Ge
Keyword(s):  
Human Cancer ◽  
The Novel
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