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BAC transgenic expression efficiency: bicistronic versus ATG-fusion strategies.

ABSTRACT: Bacterial artificial chromosome (BAC)-based transgene can be expressed bicistronically with the target gene or fused to its translation start codon. To compare the transgene expression efficiencies of these two methods, mice were created that expressed green fluorescent protein (GFP) from the genomic locus of nucleostemin using the bicistronic (NSiGFP) or the ATG-fusion approach (NSmGFP). Three lines with 1, 2, and 4 copies of the NSiGFP transgene, and two lines with 1 copy of the NSmGFP transgene were generated. Of the three NSiGFP lines, only the 4-copy line can match the NSmGFP lines in their GFP protein expression levels. Analyses of the GFP and nucleostemin RNA transcript levels exclude IRES inefficiency and suggest premature termination of the bicistronic message as the cause for low GFP expression in the NSiGFP mice. This work provides important information for designing BAC transgenics when the transgene expression level is crucial.

SUBMITTER: Lin T

PROVIDER: S-EPMC2966870 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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BAC transgenic expression efficiency: bicistronic versus ATG-fusion strategies.

Lin Tao T Yasumoto Hiroaki H Tsai Robert Y L RY

Genesis (New York, N.Y. : 2000) 20071001 10

Bacterial artificial chromosome (BAC)-based transgene can be expressed bicistronically with the target gene or fused to its translation start codon. To compare the transgene expression efficiencies of these two methods, mice were created that expressed green fluorescent protein (GFP) from the genomic locus of nucleostemin using the bicistronic (NSiGFP) or the ATG-fusion approach (NSmGFP). Three lines with 1, 2, and 4 copies of the NSiGFP transgene, and two lines with 1 copy of the NSmGFP transge ...[more]

PMID: 17941047

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Project description:p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli ?-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of ?-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3' terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in defense of cellular transformation and tumorigenesis.

Dataset Information

BAC transgenic expression efficiency: bicistronic versus ATG-fusion strategies.

Publications

BAC transgenic expression efficiency: bicistronic versus ATG-fusion strategies.

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