Limits...
Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model.

Shiue SC, Huang MZ, Tsai TF, Chang AC, Choo KB, Huang CJ, Su TS - J. Biomed. Sci. (2015)

Bottom Line: Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization.In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation.Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan. benson.shiue@gmail.com.

ABSTRACT

Background: Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.

Results: We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.

Conclusions: The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Post-transcriptional up-regulation ofASSin cells around the ventricular zone/subventricular zone (VZ/SVZ) in E14.5 embryo of theTg (ASS-Ex16-EGFP)line. (A) A frozen sagittal image of E14.5 embryo from the 16E line is shown. The sectional plane (red line) relative to the intact embryo is inserted in the bottom left corner where the embryo marking is adapted from GenePaint.org. [26] (B) Comparable region (boxed field in A) from each mouse line was selected and magnified. Red arrow points to enhanced fluorescence signals. The abbreviated mouse designations are as described in the legend to Figure 1. The labels are as follows: CP, choroid plexus; IZ, intermediate zone; NC, neocortex; OE, olfactory epithelium; OL, olfactory lobe; VZ/SVZ, ventricular zone/subventricular zone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4308890&req=5

Fig3: Post-transcriptional up-regulation ofASSin cells around the ventricular zone/subventricular zone (VZ/SVZ) in E14.5 embryo of theTg (ASS-Ex16-EGFP)line. (A) A frozen sagittal image of E14.5 embryo from the 16E line is shown. The sectional plane (red line) relative to the intact embryo is inserted in the bottom left corner where the embryo marking is adapted from GenePaint.org. [26] (B) Comparable region (boxed field in A) from each mouse line was selected and magnified. Red arrow points to enhanced fluorescence signals. The abbreviated mouse designations are as described in the legend to Figure 1. The labels are as follows: CP, choroid plexus; IZ, intermediate zone; NC, neocortex; OE, olfactory epithelium; OL, olfactory lobe; VZ/SVZ, ventricular zone/subventricular zone.

Mentions: To address the issue whether post-transcriptional regulation in ASS gene is in operation during embryonic development, EGFP expression patterns in E14.5 embryos of the Tg (ASS-Ex16-EGFP) line carrying the transcription/post-transcription couple reporter were studied. We found that the expression profiles were similar, yet significantly higher levels of EGFP expression were detected around the ventricular zone/subventricular zone (VZ/SVZ) of the brain in the Tg (ASS-Ex16-EGFP) line, i.e., 16E and 16 F, compared to that in the Tg (ASS-Ex3-EGFP) line, i.e., 3G and 3 J (Figure 3). The embryonic VZ/SVZ is the major site of neurogenesis of the mammalian telencephalon [29]. Our data suggest that there is post-transcriptional up-regulation of ASS in cells around VZ/SVZ. The mechanism and functional consequences of such a mode of regulation need to be further investigated.Figure 3


Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model.

Shiue SC, Huang MZ, Tsai TF, Chang AC, Choo KB, Huang CJ, Su TS - J. Biomed. Sci. (2015)

Post-transcriptional up-regulation ofASSin cells around the ventricular zone/subventricular zone (VZ/SVZ) in E14.5 embryo of theTg (ASS-Ex16-EGFP)line. (A) A frozen sagittal image of E14.5 embryo from the 16E line is shown. The sectional plane (red line) relative to the intact embryo is inserted in the bottom left corner where the embryo marking is adapted from GenePaint.org. [26] (B) Comparable region (boxed field in A) from each mouse line was selected and magnified. Red arrow points to enhanced fluorescence signals. The abbreviated mouse designations are as described in the legend to Figure 1. The labels are as follows: CP, choroid plexus; IZ, intermediate zone; NC, neocortex; OE, olfactory epithelium; OL, olfactory lobe; VZ/SVZ, ventricular zone/subventricular zone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308890&req=5

Fig3: Post-transcriptional up-regulation ofASSin cells around the ventricular zone/subventricular zone (VZ/SVZ) in E14.5 embryo of theTg (ASS-Ex16-EGFP)line. (A) A frozen sagittal image of E14.5 embryo from the 16E line is shown. The sectional plane (red line) relative to the intact embryo is inserted in the bottom left corner where the embryo marking is adapted from GenePaint.org. [26] (B) Comparable region (boxed field in A) from each mouse line was selected and magnified. Red arrow points to enhanced fluorescence signals. The abbreviated mouse designations are as described in the legend to Figure 1. The labels are as follows: CP, choroid plexus; IZ, intermediate zone; NC, neocortex; OE, olfactory epithelium; OL, olfactory lobe; VZ/SVZ, ventricular zone/subventricular zone.
Mentions: To address the issue whether post-transcriptional regulation in ASS gene is in operation during embryonic development, EGFP expression patterns in E14.5 embryos of the Tg (ASS-Ex16-EGFP) line carrying the transcription/post-transcription couple reporter were studied. We found that the expression profiles were similar, yet significantly higher levels of EGFP expression were detected around the ventricular zone/subventricular zone (VZ/SVZ) of the brain in the Tg (ASS-Ex16-EGFP) line, i.e., 16E and 16 F, compared to that in the Tg (ASS-Ex3-EGFP) line, i.e., 3G and 3 J (Figure 3). The embryonic VZ/SVZ is the major site of neurogenesis of the mammalian telencephalon [29]. Our data suggest that there is post-transcriptional up-regulation of ASS in cells around VZ/SVZ. The mechanism and functional consequences of such a mode of regulation need to be further investigated.Figure 3

Bottom Line: Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization.In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation.Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan. benson.shiue@gmail.com.

ABSTRACT

Background: Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.

Results: We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.

Conclusions: The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.

No MeSH data available.


Related in: MedlinePlus