Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line

2019 ◽  
Vol 61 (7-8) ◽  
pp. 393-401 ◽  
Author(s):  
Kaori Tsujino ◽  
Yuya Okuzaki ◽  
Nobuyuki Hibino ◽  
Kazuki Kawamura ◽  
Seiji Saito ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Integration Site ◽  
Chicken Line ◽  
Transgenic Chicken ◽  
Transgene Integration ◽  
Anatomical Properties

scholarly journals Whole Genome Sequencing of the Mutamouse Model Reveals Strain- and Colony-Level Variation, and Genomic Features of the Transgene Integration Site

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Matthew J. Meier ◽  
Marc A. Beal ◽  
Andrew Schoenrock ◽  
Carole L. Yauk ◽  
Francesco Marchetti
Keyword(s):  
Integration Site ◽  
Whole Genome Sequence ◽  
Comparative Genomic ◽  
Whole Genome ◽  
Missense Mutations ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
High Coverage ◽  
Deletion Event ◽  
Transgene Integration

Abstract The MutaMouse transgenic rodent model is widely used for assessing in vivo mutagenicity. Here, we report the characterization of MutaMouse’s whole genome sequence and its genetic variants compared to the C57BL/6 reference genome. High coverage (>50X) next-generation sequencing (NGS) of whole genomes from multiple MutaMouse animals from the Health Canada (HC) colony showed ~5 million SNVs per genome, ~20% of which are putatively novel. Sequencing of two animals from a geographically separated colony at Covance indicated that, over the course of 23 years, each colony accumulated 47,847 (HC) and 17,677 (Covance) non-parental homozygous single nucleotide variants. We found no novel nonsense or missense mutations that impair the MutaMouse response to genotoxic agents. Pairing sequencing data with array comparative genomic hybridization (aCGH) improved the accuracy and resolution of copy number variants (CNVs) calls and identified 300 genomic regions with CNVs. We also used long-read sequence technology (PacBio) to show that the transgene integration site involved a large deletion event with multiple inversions and rearrangements near a retrotransposon. The MutaMouse genome gives important genetic context to studies using this model, offers insight on the mechanisms of structural variant formation, and contributes a framework to analyze aCGH results alongside NGS data.

217 PRODUCTION OF A GONADOTROPIN-RELEASING HORMONE II RECEPTOR KNOCKDOWN SWINE LINE

2014 ◽  
Vol 26 (1) ◽  
pp. 222
Author(s):  
A. T. Desaulniers ◽  
R. A. Cederberg ◽  
G. A. Mills ◽  
B. R. White
Keyword(s):  
Integration Site ◽  
Gonadotropin Releasing Hormone ◽  
Injection System ◽  
Multiple Cloning Site ◽  
Sequencing Analysis ◽  
Mrna Levels ◽  
Green Fluorescence ◽  
Releasing Hormone ◽  
Transgene Integration ◽  
Shrna Sequence

Unlike the native form of gonadotropin-releasing hormone (GnRH-I), the second isoform of GnRH (GnRH-II) is highly conserved throughout evolution and is ubiquitously expressed. The pig represents one of the few species possessing coding sequence for a functional receptor specific to GnRH-II (GnRHR-II). Binding of GnRH-II to its receptor has been linked to regulation of cell proliferation, feed intake, and the interaction between energy balance and reproductive behaviour. The objective of this study was to develop a porcine model with reduced levels of endogenous GnRHR-II to examine the biological role of this G-protein coupled receptor. Previously, we produced lentiviral particles from a vector overexpressing both small hairpin RNA (shRNA) sequence specific to the porcine GnRHR-II and cDNA encoding the fluorescent ZsGreen1 protein (pLVX-shRNA2; Clontech). Transduction of swine testis cells with these particles (1.44 × 107 viral particles) reduced porcine GnRHR-II mRNA levels by 99% compared with control particles (P < 0.05). In the current study, pronuclear zygotes (n = 50) surgically collected from 1 white crossbred donor sow were microinjected into the perivitelline space with lentiviral particles containing the shRNA2 sequence (3.3 × 108 IU mL–1) using a Nikon diaphot inverted microscrope equipped with Eppendorf micromanipulators and FemtoJet injection system. A total of 40 microinjected zygotes were immediately transferred into the oviduct of 1 synchronized recipient female, resulting in the production of 5 healthy, live piglets (20% efficiency rate). Interestingly, 1 female exhibited green fluorescence, indicative of successful transgene integration and expression. Transgene integration was confirmed via conventional PCR using primers designed to amplify portions of the human U6 promoter driving the shRNA, the CMV promoter driving ZsGreen1 expression, and the multiple cloning site for incorporation of the shRNA sequence. Next, inverse PCR was performed to determine the location and number of integration sites. Sequencing analysis of PCR products revealed that a single integration site was present on chromosome 14, aligning with clone NW_003612067.1 with 99% identity and matching identities 448,946–448,37. The GnRHR-II knockdown (KD) female along with 2 female littermates were maintained and monitored during development. Attainment of puberty occurred at 149 days for the transgenic female and 145 and 151 days for littermate control gilts (P > 0.05). Upon exhibition of their third behavioural oestrus, females were bred and allowed to gestate to term. Litter size was similar between the GnRHR-II KD female (15 live piglets) and control littermates (15 and 16 live piglets). Of the 15 piglets produced, 5 (3 males and 2 females) were positive for green fluorescence, confirming germline transmission of the transgene and further evidence for a single integration site. The swine produced from this study represent the first animal model to examine the physiological implications of reduced GnRH-II receptor levels.

427 INTEGRATION SITE ANALYSIS IN TRANSGENIC PIGS BY THERMAL ASYMMETRIC INTERLACED (TAIL)-PCR AND JUNCTION PCR

2010 ◽  
Vol 22 (1) ◽  
pp. 371
Author(s):  
Q. R. Kong ◽  
Z. H. Liu
Keyword(s):  
Integration Site ◽  
Transgenic Animals ◽  
Pcr Analysis ◽  
Attractive Alternative ◽  
Transgenic Pigs ◽  
Degenerate Primers ◽  
Transgene Integration ◽  
Integration Sites ◽  
Specific Amplification ◽  
Tail Pcr

Transgenic animals have been used to study gene function, produce important proteins, xenotransplantation donor, and generate models for the study of human diseases. Recent progress in animal cloning has provided an attractive alternative to improve transgenic efficiency, through the combination of transfection and somatic cell nuclear transfer (SCNT). However, when transgenic animals are produced by SCNT using randomly transfected cells as donor, the integration sites of transgene cannot be predicted. Many methods on the basis of genome walking have been demonstrated to clone transgene integration sites but they are either complicated or inefficient. In the study, we report a PCR-based method, thermal asymmetric interlaced PCR (TAIL-PCR), which relies on a series of 3 nested PCR reactions with transgene specific, designed with melting temperature of about 64, and arbitrary degenerate primers, by control of annealing temperature to efficiently reduce the nonspecific amplification to clone the integration sites in transgenic pigs by SCNT. Junction PCR combined with transgene-specific and integration site primers was performed to confirm the integration sites. Three integration sites were found (1 mapped on chromosome 4; the other 2 met a significant match in the pig expressed sequence tag database) in 2 founder transgenic pigs. Junction PCR resulted in specific amplification bands to identify the integration sites, and segregation of the integration sites was also detected in subsequent progeny by junction PCR analysis. We also used junction PCR combining with transgene-specific 5′ and 3′ integration site primers to analyze zygosity of the integration sites. Besides the specific amplification bands amplifying by transgene specific and integration site primers, bands amplified by 5′ and 3′ integration site primers were obtained to determine the heterozygosity of integration site. In conclusion, this strategy can be efficiently employed to clone transgene integration site and determine zygosity. This work was supported by grant from the State Transgenic Research Programme of China (Grant No. 2008ZX08006-002).

Improved Production of Genetically Modified Fetuses with Homogeneous Transgene Expression After Transgene Integration Site Analysis and Recloning in Cattle

2011 ◽  
Vol 13 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Fabiana Fernandes Bressan ◽  
Moyses dos Santos Miranda ◽  
Felipe Perecin ◽  
Tiago Henrique De Bem ◽  
Flavia Thomaz Verechia Pereira ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Integration Site ◽  
Genetically Modified ◽  
Site Analysis ◽  
Transgene Integration ◽  
Integration Site Analysis

scholarly journals Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

2021 ◽  
Author(s):  
Matias Ilmari Autio ◽  
Efthymios Motakis ◽  
Arnaud Perrin ◽  
Talal Bin Amin ◽  
Zenia Tiang ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Viral Vectors ◽  
Integration Site ◽  
Embryonic Stem ◽  
Transgene Integration ◽  
Safe Harbour ◽  
Random Fashion ◽  
Endogenous Genes ◽  
Bona Fide

Stable expression of transgenes is essential in both therapeutic and research applications. Traditionally, transgene integration has been accomplished via viral vectors in a semi-random fashion, but with inherent integration site biases linked to the type of virus used. The randomly integrated transgenes may undergo silencing and more concerningly, can also lead to dysregulation of endogenous genes. Gene dysregulation can lead to malignant transformation of cells and has unfortunately given rise to cases of leukaemia in gene therapy trials. Genomic safe harbour (GSH) loci have been proposed as safe sites for transgene integration. To date, a number of sites in the human genome have been used for directed integration; however none of these pass scrutiny as bona fide GSH. Here, we conducted a computational analysis to identify 25 putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

scholarly journals Locating a transgene integration site by nanopore sequencing

2019 ◽  
Author(s):  
Peter K. Nicholls ◽  
Daniel W. Bellott ◽  
Ting-Jan Cho ◽  
Tatyana Pyntikova ◽  
David C. Page
Keyword(s):  
Germ Line ◽  
Integration Site ◽  
Cost Effective ◽  
Chromosome 9 ◽  
Biological Research ◽  
Nanopore Sequencing ◽  
Fluorescent Reporter ◽  
Transgene Integration ◽  
Integration Sites ◽  
Foreign Dna

AbstractThe introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnm (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

scholarly journals Locating and Characterizing a Transgene Integration Site by Nanopore Sequencing

2019 ◽  
Vol 9 (5) ◽  
pp. 1481-1486 ◽  
Author(s):  
Peter K. Nicholls ◽  
Daniel W. Bellott ◽  
Ting-Jan Cho ◽  
Tatyana Pyntikova ◽  
David C. Page
Keyword(s):  
Integration Site ◽  
Nanopore Sequencing ◽  
Transgene Integration

Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase

2000 ◽  
Vol 24 (6) ◽  
pp. 713-723 ◽  
Author(s):  
Rita Abranches ◽  
Ana P. Santos ◽  
Eva Wegel ◽  
Sarah Williams ◽  
Alexandra Castilho ◽  
...  
Keyword(s):  
Transgene Integration ◽  
Integration Sites

EFFECT OF SILVER NANOPARTICLES ON THE FLUORESCENCE INTENSITY OF RHODAMINE 6G AND SULFORHODAMINE 101

2016 ◽  
Vol 75 (11) ◽  
pp. 1001-1008
Author(s):  
S.V. Nikolayev ◽  
V. V. Pozhar ◽  
M. I. Dzyubenko ◽  
K. S. Nikolayev
Keyword(s):  
Silver Nanoparticles ◽  
Fluorescence Intensity ◽  
Rhodamine 6G ◽  
Sulforhodamine 101
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