scholarly journals Screening of HaCaT Clones for CCL20 Gene Knockout and Preliminary Exploration of Gene-Targeting Vector Transfection Approaches in this Cell Line

2015 ◽  
Vol 21 ◽  
pp. 21-28 ◽  
Author(s):  
Bing He
Keyword(s):  
Cell Line ◽  
Gene Targeting ◽  
Gene Knockout ◽  
Targeting Vector

94 PRODUCTION OF AN α1,3-GALACTOSYLTRANSFERASE GENE KNOCKOUT CALF USING A SINGLE TARGETING VECTOR AND REJUVENATED CELL LINES

2007 ◽  
Vol 19 (1) ◽  
pp. 164
Author(s):  
M. Urakawa ◽  
Y. Sendai ◽  
T. Sawada ◽  
Y. Shinkai ◽  
A. Ideta ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Cell Line ◽  
Southern Blot ◽  
Gene Targeting ◽  
Cell Lines ◽  
Gene Knockout ◽  
Antibiotic Resistance Gene ◽  
Blastocyst Stage ◽  
Fetal Fibroblasts ◽  
Targeting Vector

Gene targeting in large animals has the potential to be useful in agriculture. In this study, we examined whether an �1,3-galactosyltransferase-gene knockout (null) calf could be produced using a single targeting vector for disruption of both alleles and a rejuvenation of cell line by production of cloned fetuses. A promoter-less positive selection vector (pGT-22) containing an IRES (internal ribosome entry site)-antibiotic resistance gene(neo) cassette and loxP sequences was used to disrupt the bovine �1,3-galactosyltransferase (�1,3-GT) gene. In gene-targeting (GT), Japanese Black fetal fibroblasts were transfected with pGT-22, and were selected with 0.4 mg mL-1 G418. G418-resistant cells were monitored by PCR and Southern blot analysis. After PCR selection, a portion of the PCR-positive colonies were infected with the adenovirus AxCANCre, which transiently expresses Cre recombinase in the infected cells, to excise the antibiotic-resistance gene cassette (IRES/lacZ-neo) from the targeted allele. The targeted cells in which homologous recombination occurred were used for somatic cell nuclear transfer (SCNT). The cell cycle synchronization of donor cells in the early G1 phase and SCNT were performed according to the established procedure in our laboratory (Urakawa et al. 2004 Theriogenology 62, 714–728). As a result, in the first GT, one PCR-Southern blot-positive clone (0.26%, in 380 G418-resistant colony) was used for SCNT. Of 35 pulsed SCNT embryos, 4 (11.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT+/-) was established and was transfected with pGT-22 in the second GT. As a result, 8 PCR-Southern blot-positive clones (1.6%, in 508 G418-resistant colony) were recovered. The 2nd GT and the following SCNT were accomplished by the same methods used in the 1st GT and SCNT procedure. Of 36 pulsed SCNT embryos, 7 (19.4%) developed to the blastocyst stage and 3 blastocysts were nonsurgically transferred to a recipient heifer; one fetus was recovered on Day 41. The cell line (�1,3-GT-/-) was established, and was used for SCNT to obtain the �1,3-GT homozygous knockout offspring. Finally, 52 of 148 pulsed SCNT embryos (35.1%) developed to the blastocyst stage and 6 cloned embryos were transferred into 3 recipient heifers. A single knockout female offspring with normal morphology in all organs was born (Day 287) but died about one hour after birth. In this study, we established both heterozygous and homozygous �1,3-GT knockout cell lines from primary fetal fibroblasts and produced an �1,3-GT knockout offspring using a single targeting vector and rejuvenated cell lines.

scholarly journals Construction of a gene knockout CHO cell line using a simple gene targeting method

2015 ◽  
Vol 9 (Suppl 9) ◽  
pp. P2 ◽  
Author(s):  
Marina Aga ◽  
Noriko Yamano ◽  
Toshitaka Kumamoto ◽  
Jana Frank ◽  
Masayoshi Onitsuka ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Targeting ◽  
Gene Knockout ◽  
Cho Cell ◽  
Cho Cell Line

scholarly journals Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting

2017 ◽  
Vol 31 (5) ◽  
pp. 445 ◽  
Author(s):  
Li Ze ◽  
Yang Hai-Yuan ◽  
Wang Ying ◽  
Zhang Man-Ling ◽  
Liu Xiao-Rui ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Tryptophan Hydroxylase ◽  
Gene Knockout ◽  
Tryptophan Hydroxylase 2

scholarly journals A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting

2017 ◽  
Vol 19 ◽  
pp. 52-54 ◽  
Author(s):  
So-Jung Kim ◽  
Omer Habib ◽  
Jin-Soo Kim ◽  
Hyo-Won Han ◽  
Soo Kyung Koo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Line ◽  
Gene Targeting ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Stem Cell Line

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

scholarly journals MicroRNA and circRNA Expression Analysis in a Zbtb1 Gene Knockout Monoclonal EL4 Cell Line

2021 ◽  
Vol 11 ◽  
Author(s):  
Jun-Hong Wang ◽  
Chun-Wei Shi ◽  
Yi-Yuan Lu ◽  
Yan Zeng ◽  
Ming-Yang Cheng ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Gene Deletion ◽  
Cell Lymphoma ◽  
Gene Knockout ◽  
Expression Profiles ◽  
T Cell Lymphoma ◽  
Differentially Expressed ◽  
Btb Domain

Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, and a member of the mammalian Zbtb gene family. Previous studies have shown that Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by the control and gene deletion groups were determined by RNA-seq. In general, 24 differentially expressed microRNA and 16 differentially expressed circRNA were found between normal group and gene deletion group. Through further analysis of differentially expressed genes, GO term histogram and KEGG scatter plot were drawn, and three pairs of miRNA and circRNA regulatory relationships were found. This study describes the differentially expressed microRNA and circRNA in normal and Zbtb1-deficient EL4 cell lines, thus providing potential targets for drug development and clinical treatment of T-cell lymphoma.

scholarly journals Genetically Altered Mouse Models: the Good, the Bad, and the Ugly

2003 ◽  
Vol 14 (3) ◽  
pp. 154-174 ◽  
Author(s):  
Tamizchelvi Thyagarajan ◽  
Satish Totey ◽  
Mary Jo S. Danton ◽  
Ashok B. Kulkarni
Keyword(s):  
Gene Targeting ◽  
Mouse Models ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Genetically Engineered ◽  
Murine Models ◽  
Functional Roles ◽  
Targeted Gene Disruption ◽  
Gene Knockout Mouse

Targeted gene disruption in mice is a powerful tool for generating murine models for human development and disease. While the human genome program has helped to generate numerous candidate genes, few genes have been characterized for their precise in vivo functions. Gene targeting has had an enormous impact on our ability to delineate the functional roles of these genes. Many gene knockout mouse models faithfully mimic the phenotypes of the human diseases. Because some models display an unexpected or no phenotype, controversy has arisen about the value of gene-targeting strategies. We argue in favor of gene-targeting strategies, provided they are used with caution, particularly in interpreting phenotypes in craniofacial and oral biology, where many genes have pleiotropic roles. The potential pitfalls are outweighed by the unique opportunities for developing and testing different therapeutic strategies before they are introduced into the clinic. In the future, we believe that genetically engineered animal models will be indispensable for gaining important insights into the molecular mechanisms underlying development, as well as disease pathogenesis, diagnosis, prevention, and treatment.

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