Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice

P. C. Scacheri, J. S. Crabtree, E. A. Novotny, L. Garrett-Beal, A. Chen, K. A. Edgemon, S. J. Marx, A. M. Spiegel, S. C. Chandrasekharappa, F. S. Collins

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Abstract

In an effort to create a conventional knockout mouse model for multiple endocrine neoplasia type 1 (MEN1), we targeted disruption of the mouse Men1 gene through homologous recombination in ES cells. Men1 exons 2-4 were replaced by a PGK-neomycin cassette inserted in the opposite direction of Men1 transcription (Men1MSK/+). Unexpectedly, the Men1 conventional knockout was lethal in heterozygous, chimeric animals. Analysis of embryos revealed late gestational lethality with some embryos showing omphalocele. This was a very surprising phenotype, given that humans and mice that are heterozygotes for loss of function mutations in MEN1 are phenotypically normal except for a risk of endocrine tumors. Northern analysis of Men1MSK/+ embryonic stem cell RNA revealed the presence of an abundant, novel transcript of 2.1 kb, in addition to the expected wild-type transcripts of 2.7 kb and 3.1 kb. RTPCR analysis identified this aberrant transcript as arising from the antisense strand of the PGK promoter. We hypothesize that this transcript is producing either a toxic effect at the RNA level, or a dominant negative effect through the production of an amino-terminal truncated protein product. This example serves as a cautionary reminder that mouse knockouts using PGK-neo may sometimes display phenotypes that reflect more than just the loss of function of the targeted gene.

Original languageEnglish (US)
Pages (from-to)259-263
Number of pages5
JournalGenesis
Volume30
Issue number4
DOIs
StatePublished - Sep 15 2001
Externally publishedYes

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ASJC Scopus subject areas

  • Genetics
  • Endocrinology
  • Cell Biology

Cite this

Scacheri, P. C., Crabtree, J. S., Novotny, E. A., Garrett-Beal, L., Chen, A., Edgemon, K. A., Marx, S. J., Spiegel, A. M., Chandrasekharappa, S. C., & Collins, F. S. (2001). Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice. Genesis, 30(4), 259-263. https://doi.org/10.1002/gene.1072