Journal article
Insulated piggyBac and FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells
Biology open, Vol.14(10), bio061793
10/2025
DOI: 10.1242/bio.061793
PMCID: PMC12519548
PMID: 40856007
Appears in UI Libraries Support Open Access
Abstract
Transgene expression in eHAP cells, a haploid cell line commonly used to generate gene knockouts, is difficult due to its low transfection efficiency and poor expression of integrated transgenes. To enable simple and reliable transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells. We further demonstrate that transgenic haploid eHAP cells can be fused to form transgenic heterozygous diploid cells. This method creates diploid cells carrying the transgenic material of the haploid progenitors and allows for engineering of cells with defined heterozygous genotypes. These tools expand the range of experiments that can be performed in eHAP cells and other cultured cells.
Details
- Title: Subtitle
- Insulated piggyBac and FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells
- Creators
- Annabel Y. Minard - University of IowaStanley Winistorfer - University of IowaRobert C. Piper - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Biology open, Vol.14(10), bio061793
- DOI
- 10.1242/bio.061793
- PMID
- 40856007
- PMCID
- PMC12519548
- NLM abbreviation
- Biol Open
- ISSN
- 2046-6390
- eISSN
- 2046-6390
- Publisher
- Company of Biologists
- Grant note
- National Institute of General Medical Sciences: RO1GM058202 American Diabetes Association Research Foundation postdoctoral fellowshipUniversity of Iowa
This work was supported by a National Institute of General Medical Sciences grant (RO1GM058202) to R.C.P. A.Y.M. was supported by an American Diabetes Association Research Foundation postdoctoral fellowship. Open Access funding provided by University of Iowa. Deposited in PMC for immediate release.
- Language
- English
- Electronic publication date
- 08/25/2025
- Date published
- 10/2025
- Academic Unit
- Molecular Physiology and Biophysics; Anatomy and Cell Biology; Medicine Administration; Internal Medicine
- Record Identifier
- 9984949227902771
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