scholarly journals 437 DETERMINING GENE COPY NUMBER IN TRANSFECTED CAPRINE FIBROBLAST CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 375
Author(s):  
J. A. Wilson ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Dna Sequences ◽  
Copy Number ◽  
Genomic Dna ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Transgene Copy Number ◽  
Transgenic Organisms ◽  
Plasmid Construct ◽  
The Mean

Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

scholarly journals Messenger RNAs encoding the beta subunits of guinea pig (Cavia porcellus) luteinizing hormone (gpLH) and putative chorionic gonadotropin (gpCG) are transcribed from a single-copy gpLH/CGbeta gene

2001 ◽  
Vol 26 (3) ◽  
pp. 267-280 ◽  
Author(s):  
GB Sherman ◽  
DF Heilman ◽  
AJ Hoss ◽  
D Bunick ◽  
LA Lund
Keyword(s):  
Guinea Pig ◽  
Chorionic Gonadotropin ◽  
Copy Number ◽  
Genomic Dna ◽  
Gene Locus ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Beta Subunits ◽  
Coding Sequence

Neither gene locus nor gene sequence characterizations have been reported for the beta subunits of guinea pig (gp) LH and putative gp chorionic gonadotropin (CG). Descriptions of this locus would allow comparison with functionally relevant molecular genetic features of other species' homologous loci including the single-copy equid LH/CGbeta gene and the primate LHbeta-CGbeta gene cluster locus. Contiguous cDNA and genomic DNA fragments spanning the entire mature coding sequence of gpLHbeta mRNA, gpCGbeta mRNA and a homologous gpLH/CGbeta gene were amplified using PCR methodologies. With the exception of one silent mutation, the two cDNA and the genomic sequences were identical where they overlapped. Comparison of guinea pig coding sequence with LHbeta, CGbeta and LH/CGbeta sequences of other vertebrate species revealed the following order of similarity expressed as per cent coding sequence identity: rhinoceros LHbeta (83.6%)>pig LHbeta (81.8%)>donkey LH/CGbeta=bovine LHbeta (81.5%)> horse LH/CGbeta (80.6%)>dog LHbeta (79.7%)>human LHbeta (78.2%)>rat LHbeta (77.9%)>human CGbeta (75.8%)>turkey LHbeta (52.7%); values that are generally consistent with recently postulated phylogenetic relationships. Like the consensus mammalian LHbeta gene, the 5'-flanking region of the gpLH/CGbeta gene contains a single TATA sequence 37 bp upstream of the translation start codon. The first in-frame stop codon occurred at codon position +122 which is consistent with the 121 amino acid residue length of the consensus mammalian mature LHbeta peptide. To estimate gene copy number, full-length gpLHbeta cDNA was radiolabeled and hybridized to Southern blots of guinea pig genomic DNA digested with a panel of six restriction endonucleases. The resulting simple hybridization pattern strongly suggested that there is a single-copy gpLH/CGbeta gene. Northern analysis of total pituitary RNA using the same probe indicated that gpLHbeta transcript size is indistinguishable from that of consensus mammalian pituitary LHbeta mRNAs ( approximately 750 nucleotides). Despite amplifying gpCGbeta from placental RNA, positive signal was not detected in Northern blot lanes containing guinea pig total RNA prepared from placentae collected at three gestational ages (17.3 days, 24.3 days and 68 days (term)). Other data suggest that inability to detect Northern blot signal could have been due to low relative tissue concentrations of gpCGbeta transcript and/or sampling at gestational time-points that missed peak periods of mRNA expression. We conclude that, with respect to gene copy number, coding sequence and pituitary mRNA size, the gpLH/CGbeta gene locus reflects the CTP-less consensus mammalian LHbeta condition. However, based on the capacity of this single-copy gene to express in both pituitary and placental tissues, gpLH/CGbeta also exhibits functional similarities with the single-copy equine LH/CGbeta locus.

scholarly journals Digital PCR (dPCR) and qPCR Mediated Determination of Transgene Copy Number in the Forage Legume White Clover (Trifolium Repens).

2021 ◽  
Author(s):  
Rafael Narancio ◽  
Ulrik John ◽  
John Mason ◽  
Paula Giraldo ◽  
German Spangenberg
Keyword(s):  
White Clover ◽  
Reference Genes ◽  
Copy Number ◽  
Gene Copy Number ◽  
Single Copy ◽  
Digital Pcr ◽  
Gene Copy ◽  
Relative Quantification ◽  
Transgene Copy Number ◽  
Copy Number Estimation

Abstract Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

scholarly journals Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

2000 ◽  
Vol 97 (1) ◽  
pp. 222-227 ◽  
Author(s):  
J. Herrick ◽  
X. Michalet ◽  
C. Conti ◽  
C. Schurra ◽  
A. Bensimon
Keyword(s):  
Fluorescent Probe ◽  
Copy Number ◽  
Genomic Dna ◽  
Single Gene ◽  
Gene Copy Number ◽  
Gene Copy

scholarly journals Endogenous RNA interference is driven by copy number

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Cruz ◽  
Jonathan Houseley
Keyword(s):  
Rna Interference ◽  
Transposable Elements ◽  
Copy Number ◽  
Defense Mechanism ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Protein Coding ◽  
Endogenous Loci ◽  
Pervasive Transcription

A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome.

scholarly journals Real-time PCR for the detection of precise transgene copy number in durum wheat

2011 ◽  
Vol 16 (4) ◽  
Author(s):  
Agata Gadaleta ◽  
Angelica Giancaspro ◽  
Maria Cardone ◽  
Antonio Blanco
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Gene Copy Number ◽  
Correlation Coefficients ◽  
Gene Copy ◽  
Transgene Copy Number ◽  
Locus Number ◽  
Wheat Lines

AbstractRecent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

Phenotypic and genotypic characteristics of epidermal growth factor receptor (EGFR) in colorectal cancer patients

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 4110-4110
Author(s):  
G. A. Milano ◽  
M. C. Etienne-Grimaldi ◽  
M. Francoual ◽  
D. Benchimol ◽  
M. Chazal ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Copy Number ◽  
Binding Assay ◽  
Gene Copy Number ◽  
Normal Mucosa ◽  
Gene Copy ◽  
Colorectal Tumors ◽  
Egfr Gene ◽  
Egfr Expression ◽  
The Mean

4110 Background: Colorectal tumors express EGFR and are responsive to anti-EGFR therapies. However, there is no tumoral predictive factor for anti-EGFR therapy in colorectal cancer and EGFR gene copy number is currently a good candidate. The aim of this study was to examine relationships between EGFR germinal polymorphisms, EGFR gene copy number and EGFR expression. Methods: Eighty primary colorectal tumors were analyzed along with 39 normal mucosas. Tumor staging was : 4 stage 0, 13 stage I, 22 stage II, 23 stage III and 18 stage IV. EGFR -216G>T and -191C>A genotypes were analyzed by PCR-RFLP, CA-repeats polymorphism in intron 1 by fluorescent genotyping and gene copy number was measured by PCR amplification. EGFR expression was quantified with the reference Scatchard binding assay giving high- and low-affinity sites along with Kd values (Francoual M et al. Ann Oncol 17, 2006). Results: The number of CA- repeats varied from 14 to 21. Considered genotypes were superimposable between tumoral and normal tissues. A linkage disequilibrium was noted between -216G>T and -191C>A genotypes (p = 0.011). CA-repeats polymorphism and -216G>T genotype were not independent (p = 0.002). No relationship was observed between any of the analyzed EGFR genotypes and EGFR expression. EGFR expression was not related to gene amplification. EGFR gene amplification in tumor and normal tissue varied over a 4.7- and 2.9-fold range, respectively, and were not correlated. The mean value of the tumor/normal mucosa amplification ratio was 1.16 (range 0.55–2.68) and 14% of patients exhibited lower amplification in the tumor relative to the normal mucosa (ratio < 0.75). The mean ratio of high-affinity sites between tumor and normal mucosa was 1.20 (range 0.03–13.33). Conclusions: In colorectal tumors, neither EGFR gene amplification nor EGFR germinal gene polymorphisms influenced EGFR expression quantified with a specific ligand-binding assay. No significant financial relationships to disclose.

scholarly journals Why haploinsufficiency persists

2019 ◽  
pp. 201900437 ◽  
Author(s):  
Summer A. Morrill ◽  
Angelika Amon
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Data Set ◽  
Cellular Processes ◽  
Sensitivity Data ◽  
Cell To Cell Variability ◽  
Decrease Gene Expression

Haploinsufficiency describes the decrease in organismal fitness observed when a single copy of a gene is deleted in diploids. We investigated the origin of haploinsufficiency by creating a comprehensive dosage sensitivity data set for genes under their native promoters. We demonstrate that the expression of haploinsufficient genes is limited by the toxicity of their overexpression. We further show that the fitness penalty associated with excess gene copy number is not the only determinant of haploinsufficiency. Haploinsufficient genes represent a unique subset of genes sensitive to copy number increases, as they are also limiting for important cellular processes when present in one copy instead of two. The selective pressure to decrease gene expression due to the toxicity of overexpression, combined with the pressure to increase expression due to their fitness-limiting nature, has made haploinsufficient genes extremely sensitive to changes in gene expression. As a consequence, haploinsufficient genes are dosage stabilized, showing much more narrow ranges in cell-to-cell variability of expression compared with other genes in the genome. We propose a dosage-stabilizing hypothesis of haploinsufficiency to explain its persistence over evolutionary time.

Enhanced Detection of DNA Sequences Using End-Point PCR Amplification and Online Gel Electrophoresis (GE)-ICP-MS: Determination of Gene Copy Number Variations

2014 ◽  
Vol 86 (22) ◽  
pp. 11028-11032 ◽  
Author(s):  
T. Iglesias González ◽  
M. Espina ◽  
L. M. Sierra ◽  
J. Bettmer ◽  
E. Blanco-González ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Gel Electrophoresis ◽  
Copy Number ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Copy Number Variations ◽  
Gene Copy ◽  
End Point ◽  
Icp Ms

Analysis of IGF1R gene copy number by silver in situ hybridization (SISH) in non-small cell lung cancer (NSCLC)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7525-7525
Author(s):  
M. W. Wynes ◽  
S. Singh ◽  
R. Dziadziuszko ◽  
K. Dziadziuszko ◽  
K. Jaskiewicz ◽  
...  
Keyword(s):  
Copy Number ◽  
Statistical Difference ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Medical Systems ◽  
Gene Copies ◽  
Potential Biomarker ◽  
Significant Difference ◽  
The Mean ◽  
Pathological Stages

7525 Background: IGF1R is a potential biomarker for predicting outcome of NSCLC patients (pts) treated with therapeutics targeting the IGF1R signaling pathway. SISH can be expeditiously analyzed on a bright field microscope given that the morphology of the tissue is observed concurrent to the scoring of discrete gene signals. Methods: An experimental SISH probe designed by Ventana Medical Systems (Tucson, AZ) was used to evaluate IGF1R gene copy number on a tissue microarray containing triplicate samples from 189 pts surgically treated for NSCLC (median follow-up 4 years and 5-year survival probability of 40%). Valid results, at least one core with sufficient tumor content for assessment, were obtained for 166 patients. There were 128 males; 93 squamous cell carcinomas (SCC), 47 adenocarcinomas, 5 large cell carcinomas and 21 other histologies. The pathological stages were I: 68, II: 37 and III/IV: 60. The mean number of IGF1R gene copies/nuclei/core was determined by a board certified pathologist counting 50 nuclei in each core. The mean number of IGF1R gene copies/nuclei/patient was determined by using, within the triplicates, the core with the highest gene copy number/nuclei. Results: The mean number of IGF1R gene copies/nuclei per patient was 2.26 (range: 1.12 - 7.56; standard deviation 0.81). The median copy number was 2.11. The pts were divided into two groups, those with 2.1 genes/nuclei or less and those with greater than 2.1 genes/nuclei. There was no statistical difference related to gender (p=0.422) or between pathological stages, (p=0.221). However, there was a highly significant difference between the two categories when considering histological pattern. Among patients with SCC, 66.3% had high copy number compared to 33.7 % in non-SCC histologies (p=0.008). Analysis of overall survival comparing pts with low vs. high IGF1R gene copy number revealed no statistical difference in the median survival: 1.5 yrs (95% CI 0–3.7 yrs) vs. 3.3 yrs (2.0–4.5 yrs) or the 3-year survival: 46% (35–57%) vs. 52% (41–63%). Conclusions: IGF1R gene copy number detected by SISH is higher in SCC than in other histologies of NSCLC, but does not associate with gender, pathological stage or survival. IGF1R SISH should be further explored as a predictive biomarker for IGF1R therapeutics. [Table: see text]

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