Derivation of the clonal-cell lines from Siberian sturgeon (Acipenser baerii ) head-kidney cell lines and its applicability to foreign gene expression and virus culture

2018 ◽  
Vol 92 (5) ◽  
pp. 1273-1289 ◽  
Author(s):  
J. H. Ryu ◽  
M. S. Kim ◽  
J. H. Kang ◽  
D. H. Kim ◽  
Y. K. Nam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Kidney Cell ◽  
Head Kidney ◽  
Foreign Gene ◽  
Foreign Gene Expression ◽  
Acipenser Baerii ◽  
Clonal Cell ◽  
Clonal Cell Lines ◽  
Virus Culture

scholarly journals Reduction of Target Gene Expression by a Modified U1 snRNA

2001 ◽  
Vol 21 (8) ◽  
pp. 2815-2825 ◽  
Author(s):  
S. A. Beckley ◽  
P. Liu ◽  
M. L. Stover ◽  
S. I. Gunderson ◽  
A. C. Lichtler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Stable Transfection ◽  
Rnase Protection ◽  
Intact Cells ◽  
Clonal Cell ◽  
U1 Snrna ◽  
Clonal Cell Lines

ABSTRACT Although the primary function of U1 snRNA is to define the 5′ donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5′ donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], β-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3′ cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.

scholarly journals Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 549-549
Author(s):  
Jason H. Rogers ◽  
Rohit Gupta ◽  
Jaime M. Reyes ◽  
Lorenzo Brunetti ◽  
Michael C. Gundry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Sanger Sequencing ◽  
Lymphoblastic Leukemia ◽  
Clonal Cell ◽  
Ribonucleoprotein Complexes ◽  
Clonal Cell Lines ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models. We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D). We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/-clonal cell lines are distinct from their isogenic IKZF1-/-counterparts. Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/-cells and IKZF1IK6/+cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. Disclosures No relevant conflicts of interest to declare.

scholarly journals Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells

2003 ◽  
Vol 178 (1) ◽  
pp. 61-69 ◽  
Author(s):  
JA Stirland ◽  
ZC Seymour ◽  
S Windeatt ◽  
AJ Norris ◽  
P Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cell Lines ◽  
Promoter Activity ◽  
Expression Patterns ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Clonal Cell ◽  
Clonal Cell Lines

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.

ESTABLISHMENT AND CHARACTERIZATION OF CLONAL CELL LINES FROM THE VAGINA OF p53-DEFICIENT YOUNG MICE

2002 ◽  
Vol 38 (10) ◽  
pp. 547 ◽  
Author(s):  
KAYO TANAHASHI ◽  
SHINOBU SHIBAHARA ◽  
MINAKO OGAWA ◽  
MAKOTO HANAZONO ◽  
SHINICHI AIZAWA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Clonal Cell ◽  
Clonal Cell Lines ◽  
Young Mice

Implantation of c-mycERTAMImmortalized Human Mesencephalic-Derived Clonal Cell Lines Ameliorates Behavior Dysfunction in a Rat Model of Parkinson’s Disease

2009 ◽  
Vol 18 (2) ◽  
pp. 307-320 ◽  
Author(s):  
Erik A. Miljan ◽  
Susan J. Hines ◽  
Priyadarshini Pande ◽  
Randolph L. Corteling ◽  
Caroline Hicks ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Rat Model ◽  
Clonal Cell ◽  
Clonal Cell Lines

High Levels of Foreign Gene Expression in Hepatocytes after Tail Vein Injections of Naked Plasmid DNA

1999 ◽  
Vol 10 (10) ◽  
pp. 1735-1737 ◽  
Author(s):  
Guofeng Zhang ◽  
Vladimir Budker ◽  
Jon A. Wolff
Keyword(s):  
Gene Expression ◽  
Plasmid Dna ◽  
Foreign Gene ◽  
Foreign Gene Expression ◽  
Tail Vein ◽  
Naked Plasmid ◽  
Naked Plasmid Dna
Sign in / Sign up

Export Citation Format

Share Document