Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells.
BACKGROUND
Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells.


RESULTS
A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene.


CONCLUSIONS
IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.
Department of Cell Biology and Physiology, Pittsburgh Development Center, Magee-Women's Research Institute, University of Pittsburgh School of Medicine, PA 15261, USA. daiutol@upmc.edu
Leonardo
L
D'Aiuto
Clinton S
CS
Robison
Margherita
M
Gigante
Edward
E
Nwanegbo
Benjamin
B
Shaffer
Meena
M
Sukhwani
Carlos A
CA
Castro
J Richard
JR
Chaillet
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000502:physiology*; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D018664:Interleukin-12