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

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

PCR-based approaches for identification of multi-copy transgene integration sites in mouse genome

2006 ◽  
Vol 51 (18) ◽  
pp. 2231-2235 ◽  
Author(s):  
Xudong Zhao ◽  
Suying Dang ◽  
Bin Liang ◽  
Xia Lei ◽  
Zheng Chen ◽  
...  
Keyword(s):  
Mouse Genome ◽  
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

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.

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

408 FOREIGN GENE INTEGRATION PATTERNS IN TRANSGENIC PORCINE FETUSES PRODUCED BY ICSI-MEDIATED GENE TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 319
Author(s):  
H. Saito ◽  
M. Kurome ◽  
R. Tomii ◽  
S. Ueno ◽  
K. Hiruma ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Southern Blot ◽  
Disease Model ◽  
Foreign Gene ◽  
Fish Analysis ◽  
Pronuclear Microinjection ◽  
Model Study ◽  
Integration Sites ◽  
Random Site

We previously reported that transgenic (TG) pigs can be produced from in vitro-matured oocytes using intracytoplasmic sperm injection-mediated gene transfer (ICSI-mediated method) (Kurome et al. 2006 Transgenic Res. 15, 229–240). We subsequently studied the expression of a foreign gene which had been introduced by the ICSI-mediated method. We found that the ICSI-mediated method is considerably less likely than the pronuclear microinjection method to produce embryos in which transgene-positive and transgene-negative cells co-exist, that is, mosaic embryos (Saito et al. 2006 Reprod. Fertil. Dev. 18, 297 abst). Therefore, in order to further investigate the ICSI-mediated method, the present study was conducted to address the integration patterns of foreign genes introduced by this method. In particular, we wished to determine the number of transgene copies and number of chromosomal integration sites. TG pig fetuses, obtained by the ICSI-mediated method in a separate cardiac disease model study, were used in the present study. Porcine cumulus-oocyte complexes that had been collected from slaughterhouse ovaries were subjected to in vitro maturation in NCSU23 medium to produce MII oocytes to be used in this study. Porcine spermatozoa frozen in Beltsville Thawing Solution (BTS) were thawed rapidly in a 37�C water bath, and each spermatozoon was decapitated using ultrasound (28 kHz, 100 W; W-113; Honda Electronics Co., Ltd, Aichi, Japan). The heads (2 to 5 � 105/10 �L) were co-incubated with 2.5 ng �L-1 of rabbit calreticulin cDNA (�MHC-CRT-HA: 7.5 kb) for five min at room temperature, and then microinjected into MII oocytes using a piezo-micromanipulator. An electric stimulus (DC 150 V mm-1, 100 �s) was applied 10 to 40 min after microinjection in order to activate the oocytes. The embryos were cultured in PZM-5 medium for one to two days, and then transferred into the oviducts of recipient gilts, whose estrous cycle had been synchronized using 1000 IU eCG and 1500 IU hCG. Fetuses were collected 33 or 50 days later, and a primary cell line (fibroblast) was established. For each fetus, the number of transgene copies was determined by Southern blot. In addition, the chromosomal sites, where the foreign gene had integrated, were identified, and the number of integration sites was determined by fluoresent in situ hybridization (FISH). A total of 454 ICSI embryos were transferred to 4 recipients (92 to 135 embryos/recipient). All recipients became pregnant and 23 fetuses (5.1%, 23/454), including 7 TG fetuses (30.4%, 7/23), were obtained. Southern blot analysis showed that the number of transgene copies varied between 1 and 300 (1 copy: 1 fetus; 10 copies: 2 fetuses; 30 copies: 3 fetuses; 300 copies: 1 fetus). FISH analysis showed that in TG fetuses, the foreign gene had integrated at only a single chromosomal site, and this site varied from TG fetus to TG fetus. These results demonstrate that, in the case of ICSI-mediated gene transfer, as is the case for gene transfer by pronuclear microinjection, the integration patterns are: multiple copy, random site, and single site integration. This study was supported by PROBRAIN.

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