Identifying and genotyping transgene integration loci

Zhong Liang, Amy Marie Breman, Brenda Grimes, Elliot Rosen

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.

Original languageEnglish
Pages (from-to)979-983
Number of pages5
JournalTransgenic Research
Volume17
Issue number5
DOIs
StatePublished - Oct 2008

Fingerprint

Transgenes
genotyping
transgenes
loci
mice
Genetic Markers
Transgenic Mice
genetically modified organisms
DNA Ligases
Phenotype
Biological Phenomena
genomics
phenotype
Chromosomes, Human, Pair 14
Factor X
genetic markers
annealing
Insertional Mutagenesis
Gene Silencing
DNA

Keywords

  • Factor X
  • FISH
  • Genotyping
  • Insertion site
  • Inverse PCR
  • Transgene

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Biotechnology
  • Food Science

Cite this

Identifying and genotyping transgene integration loci. / Liang, Zhong; Breman, Amy Marie; Grimes, Brenda; Rosen, Elliot.

In: Transgenic Research, Vol. 17, No. 5, 10.2008, p. 979-983.

Research output: Contribution to journalArticle

Liang, Zhong ; Breman, Amy Marie ; Grimes, Brenda ; Rosen, Elliot. / Identifying and genotyping transgene integration loci. In: Transgenic Research. 2008 ; Vol. 17, No. 5. pp. 979-983.
@article{7a9d7250101244ccbedccf58fb17d92e,
title = "Identifying and genotyping transgene integration loci",
abstract = "The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.",
keywords = "Factor X, FISH, Genotyping, Insertion site, Inverse PCR, Transgene",
author = "Zhong Liang and Breman, {Amy Marie} and Brenda Grimes and Elliot Rosen",
year = "2008",
month = "10",
doi = "10.1007/s11248-008-9190-7",
language = "English",
volume = "17",
pages = "979--983",
journal = "Transgenic Research",
issn = "0962-8819",
publisher = "Springer Netherlands",
number = "5",

}

TY - JOUR

T1 - Identifying and genotyping transgene integration loci

AU - Liang, Zhong

AU - Breman, Amy Marie

AU - Grimes, Brenda

AU - Rosen, Elliot

PY - 2008/10

Y1 - 2008/10

N2 - The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.

AB - The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.

KW - Factor X

KW - FISH

KW - Genotyping

KW - Insertion site

KW - Inverse PCR

KW - Transgene

UR - http://www.scopus.com/inward/record.url?scp=50849096679&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=50849096679&partnerID=8YFLogxK

U2 - 10.1007/s11248-008-9190-7

DO - 10.1007/s11248-008-9190-7

M3 - Article

VL - 17

SP - 979

EP - 983

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 5

ER -