Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice.
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 (Men1(MSK/+)). 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 Men1(MSK/+) 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. RT-PCR 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.
Scacheri
P C
PC
National Human Genome Research Institute, National Institute of Health, Bethesda, Maryland 20892, USA.
Crabtree
J S
JS
Novotny
E A
EA
Garrett-Beal
L
L
Chen
A
A
Edgemon
K A
KA
Marx
S J
SJ
Spiegel
A M
AM
Chandrasekharappa
S C
SC
Collins
F S
FS
D000818:Animals; D015153:Blotting, Western; D002678:Chimera / Q000235:genetics; D020964:Embryo Loss / Q000235:genetics*; D004622:Embryo, Mammalian / Q000378:metabolism; D005091:Exons / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods; D005799:Genes, Dominant / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006554:Hernia, Umbilical / Q000235:genetics; D006579:Heterozygote; D051379:Mice; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009355:Neomycin / Q000096:biosynthesis; D009363:Neoplasm Proteins / Q000235:genetics*; D010641:Phenotype; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
C105373:MEN1 protein, human; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D009355:Neomycin