Journal article
Transgenic Xenopus laevis embryos can be generated using ϕC31 integrase
Nature methods, Vol.2(12), pp.975-979
12/2005
DOI: 10.1038/nmeth814
PMCID: PMC3552317
PMID: 16299484
Abstract
Phage ϕC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA
1
. Here we show ϕC31 integrase can be used to generate transgenic
Xenopus laevis
embryos. mRNA encoding integrase was co-injected with a reporter plasmid containing a CMV promoter driven GFP into one cell embryos. The reporter plasmid was integrated into the genome. GFP expression, though robust, was in a limited number of tissues and varied among the embryos analyzed. We attributed this restriction to transcriptional silencing by chromosome position effects. Modification of the reporter plasmid by bracketing the CMV-GFP region with tandem copies of the chicken β-globin 5′ HS4 insulator
2
relieved position effects. Embryos transgenic with insulated CMV-GFP expressed GFP uniformly. Tissue specific expression was achieved when the CMV promoter was replaced with a 551 base-pair minimal gamma crystallin lens promoter from
Xenopus.
Embryos transgenic with this plasmid had GFP expression limited to the lens of the eye. We observed that about a third of embryos assayed one week after fertilization were transgenic. These experiments demonstrate that the integration of insulated gene sequences using ϕC31 integrase can be used to efficiently create transgenic embryos in
Xenopus laevis
and may increase the practical use of ϕC31 integrase in other systems as well.
Details
- Title: Subtitle
- Transgenic Xenopus laevis embryos can be generated using ϕC31 integrase
- Creators
- Bryan G AllenDaniel L Weeks
- Resource Type
- Journal article
- Publication Details
- Nature methods, Vol.2(12), pp.975-979
- DOI
- 10.1038/nmeth814
- PMID
- 16299484
- PMCID
- PMC3552317
- NLM abbreviation
- Nat Methods
- ISSN
- 1548-7091
- eISSN
- 1548-7105
- Grant note
- R01 GM069944-05A2 || GM / National Institute of General Medical Sciences : NIGMS
- Language
- English
- Date published
- 12/2005
- Academic Unit
- Stead Family Department of Pediatrics; Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984025293302771
Metrics
25 Record Views