Transcriptional Properties of Genomic Transgene Integration Sites Marked by Electroporation or Retroviral Infection†

Biochemistry ◽  
2000 ◽  
Vol 39 (24) ◽  
pp. 7041-7049 ◽  
Author(s):  
Alexandra Baer ◽  
Dirk Schübeler ◽  
Jürgen Bode
Keyword(s):  
Retroviral Infection ◽  
Transgene Integration ◽  
Integration Sites

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

FISH Analysis of 142 EGFP Transgene Integration Sites into the Mouse Genome

Genomics ◽  
2002 ◽  
Vol 80 (6) ◽  
pp. 564-574 ◽  
Author(s):  
Tomoko Nakanishi ◽  
Asato Kuroiwa ◽  
Shuichi Yamada ◽  
Ayako Isotani ◽  
Atsuko Yamashita ◽  
...  
Keyword(s):  
Mouse Genome ◽  
Fish Analysis ◽  
Transgene Integration ◽  
Integration Sites

Association of transgene integration sites with chromosome rearrangements in hexaploid oat

2000 ◽  
Vol 100 (6) ◽  
pp. 872-880 ◽  
Author(s):  
S. Svitashev ◽  
E. Ananiev ◽  
W. P. Pawlowski ◽  
D. A. Somers
Keyword(s):  
Chromosome Rearrangements ◽  
Transgene Integration ◽  
Integration Sites

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).

scholarly journals RAISING: a high-performance method for identifying random transgene integration sites

2021 ◽  
Author(s):  
Yusaku Wada ◽  
Tomoo Sato ◽  
Hiroo Hasegawa ◽  
Takahiro Matsudaira ◽  
Naganori Nao ◽  
...  
Keyword(s):  
High Performance ◽  
Viral Vectors ◽  
Viral Infections ◽  
Therapeutic Interventions ◽  
Transgene Integration ◽  
Adult T Cell Leukemia ◽  
Integration Sites ◽  
Infected Cells ◽  
Novel Method ◽  
Rapid Amplification

Abstract Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

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.

Fish analysis of multiple transgene integration sites in a beta casein-antithrombin III goat line

1998 ◽  
Vol 49 (1) ◽  
pp. 398 ◽  
Author(s):  
J. Williams ◽  
F.A.Ponce de Leon ◽  
P. Midura ◽  
M. Hamngton ◽  
H. Meade ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Fish Analysis ◽  
Transgene Integration ◽  
Integration Sites ◽  
Beta Casein

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

2008 ◽  
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

scholarly journals Antiretroviral APOBEC3 Cytidine Deaminases Alter HIV-1 Provirus Integration Site Profiles

2019 ◽  
Author(s):  
Hannah O. Ajoge ◽  
Tyler M. Renner ◽  
Kasandra Bélanger ◽  
Hinissan P. Kohio ◽  
Macon D. Coleman ◽  
...  
Keyword(s):  
Integration Site ◽  
Chromosomal Dna ◽  
Retroviral Infection ◽  
Cytidine Deaminases ◽  
Gene Coding ◽  
Integration Sites ◽  
Site Targeting ◽  
Provirus Integration Site ◽  
Hiv Integration ◽  
Hiv 1

ABSTRACTAPOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. While the catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total restriction of infection, sublethal levels of deamination contribute to the genetic evolution of HIV-1. So far, little is known about how A3 might impact HIV-1 integrations into human chromosomal DNA. Using a deep sequencing approach, we analyzed the influence A3F and A3G on HIV-1 integration site selections. DNA editing was detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 enzymes decreased insertions into gene coding sequences and increased integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicate A3 as host factors that influence HIV-1 integration site selection and promote insertions into genomic sites that are transcriptionally less active.GRAPHICAL ABSTRACTSchematic depicting the influence of APOBEC3 (A3) proteins on HIV integration site targeting.Left, in the absence of A3, HIV has a strong preference for integrating into genes. Right, both catalytic active and non-catalytic A3 mutants decrease integration into genes and increase integration into SINE elements and in transcription-silencing non-B DNA features.

scholarly journals Screen and Verification for Transgene Integration Sites in Pigs

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Linyuan Ma ◽  
Yuzhe Wang ◽  
Haitao Wang ◽  
Yiqing Hu ◽  
Jingyao Chen ◽  
...  
Keyword(s):  
Transgene Integration ◽  
Integration Sites
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