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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

Analysis of the transgene structure in the BAC (ASS-EGFP) transgenic mice. (A) Overall view of BAC (ASS-EGFP) constructs showing relative positions of the EGFP transgene and the human ASS exons. Insertions of the EGFP gene with a polyadenylation signal into exon 3 (EGFP-pA) or an IRES-EGFP sequence into exon 16 of the ASS gene of BAC clone RP11-52B13 created the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) constructs, respectively. The lengths of the 5′ and 3′ human genomic sequences included in the BAC construct and the ASS structural gene are shown. Wavy line at the end represents the vector sequence. (B) Determination of transgene copy number. Genomic DNAs from offspring of the second generation transgenic mice (F2) was digested with EcoRI and Southern blot analysis was performed using an EGFP probe. The horizontal grey arrows are schematic representation of the transgene; the green bars represent the EGFP sequence; downward arrows indicate the EcoRI sites shown with expected sizes of the hybridized EcoRI fragments. The signal of a BAC (ASS-Ex3-EGFP) DNA preparation with predetermined copy number was used to estimate the transgene copy number in the mouse lines. The abbreviated transgenic line designations 3G, 3 J and 16E, 16H were two different lines each from the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) transgenes, respectively; the abbreviated designations are also used in other figures. (C) Confirmation of head-to-tail transgene integration. The PstI sites (downward arrows) and the expected 1,044-bp head-to-tail hybridized fragments are shown. The size of junction fragment may be greater than 939 bp (>939 bp) which is determined by position of the next PstI site in the mouse genome. (D) Monitoring of ASS-EGFP transgene transmission by fluorescence signals of tail samples. Pedigree analysis of the transgenic line Tg (ASS-Ex16-EGFP) 16 F is shown. Upper and lower rows were observed by dissecting microscopy under fluorescent light or white light, respectively.
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Fig1: Analysis of the transgene structure in the BAC (ASS-EGFP) transgenic mice. (A) Overall view of BAC (ASS-EGFP) constructs showing relative positions of the EGFP transgene and the human ASS exons. Insertions of the EGFP gene with a polyadenylation signal into exon 3 (EGFP-pA) or an IRES-EGFP sequence into exon 16 of the ASS gene of BAC clone RP11-52B13 created the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) constructs, respectively. The lengths of the 5′ and 3′ human genomic sequences included in the BAC construct and the ASS structural gene are shown. Wavy line at the end represents the vector sequence. (B) Determination of transgene copy number. Genomic DNAs from offspring of the second generation transgenic mice (F2) was digested with EcoRI and Southern blot analysis was performed using an EGFP probe. The horizontal grey arrows are schematic representation of the transgene; the green bars represent the EGFP sequence; downward arrows indicate the EcoRI sites shown with expected sizes of the hybridized EcoRI fragments. The signal of a BAC (ASS-Ex3-EGFP) DNA preparation with predetermined copy number was used to estimate the transgene copy number in the mouse lines. The abbreviated transgenic line designations 3G, 3 J and 16E, 16H were two different lines each from the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) transgenes, respectively; the abbreviated designations are also used in other figures. (C) Confirmation of head-to-tail transgene integration. The PstI sites (downward arrows) and the expected 1,044-bp head-to-tail hybridized fragments are shown. The size of junction fragment may be greater than 939 bp (>939 bp) which is determined by position of the next PstI site in the mouse genome. (D) Monitoring of ASS-EGFP transgene transmission by fluorescence signals of tail samples. Pedigree analysis of the transgenic line Tg (ASS-Ex16-EGFP) 16 F is shown. Upper and lower rows were observed by dissecting microscopy under fluorescent light or white light, respectively.

Mentions: To investigate transcriptional and post-transcriptional regulation of the ASS gene, we established a transgenic mouse system using a modified bacterial artificial chromosome (BAC) carrying the human ASS gene tagged with the enhanced green fluorescent protein (EGFP) reporter gene [20]. Two transgenic mouse lines were generated. One line was Tg (ASS-Ex3-EGFP) which carries the transcription reporter BAC (ASS-Ex3-EGFP) (Figure 1A), where EGFP was knocked-in at the initiation codon of the human ASS gene and EGFP transcription is terminated by a SV40 poly (A) signal. EGFP activities of Tg (ASS-Ex3-EGFP) mainly reflect promoter activities of the ASS gene. Another line Tg (ASS-Ex16-EGFP) carries the transcription/post-transcription couple reporter BAC (ASS-Ex16-EGFP) (Figure 1A), where EGFP with an internal ribosome entry site (IRES) is inserted into the terminal exon at site between the stop codon and the polyA signal of the ASS gene; in such a configuration, translation of EGFP in the bicistronic transcript is regulated by the IRES mechanism. EGFP activities thus expressed are subjected to both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. Using these transgenic mouse lines, we have taken liver, the organ for urea production, intestine and kidney that are responsible for arginine biosynthesis as a model for temporal and spatial expression analyses [20]. We found the expression of the EGFP reporter gene in the transgenic mice faithfully reproduced that of the endogenous gene, suggesting that sufficient ASS regulatory elements are included in the transgene. Moreover, comparison between the EGFP expression profiles of the two transgenic lines indicated the developmental and tissue-specific regulation is mainly controlled at the transcriptional level [20].Figure 1


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)

Analysis of the transgene structure in the BAC (ASS-EGFP) transgenic mice. (A) Overall view of BAC (ASS-EGFP) constructs showing relative positions of the EGFP transgene and the human ASS exons. Insertions of the EGFP gene with a polyadenylation signal into exon 3 (EGFP-pA) or an IRES-EGFP sequence into exon 16 of the ASS gene of BAC clone RP11-52B13 created the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) constructs, respectively. The lengths of the 5′ and 3′ human genomic sequences included in the BAC construct and the ASS structural gene are shown. Wavy line at the end represents the vector sequence. (B) Determination of transgene copy number. Genomic DNAs from offspring of the second generation transgenic mice (F2) was digested with EcoRI and Southern blot analysis was performed using an EGFP probe. The horizontal grey arrows are schematic representation of the transgene; the green bars represent the EGFP sequence; downward arrows indicate the EcoRI sites shown with expected sizes of the hybridized EcoRI fragments. The signal of a BAC (ASS-Ex3-EGFP) DNA preparation with predetermined copy number was used to estimate the transgene copy number in the mouse lines. The abbreviated transgenic line designations 3G, 3 J and 16E, 16H were two different lines each from the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) transgenes, respectively; the abbreviated designations are also used in other figures. (C) Confirmation of head-to-tail transgene integration. The PstI sites (downward arrows) and the expected 1,044-bp head-to-tail hybridized fragments are shown. The size of junction fragment may be greater than 939 bp (>939 bp) which is determined by position of the next PstI site in the mouse genome. (D) Monitoring of ASS-EGFP transgene transmission by fluorescence signals of tail samples. Pedigree analysis of the transgenic line Tg (ASS-Ex16-EGFP) 16 F is shown. Upper and lower rows were observed by dissecting microscopy under fluorescent light or white light, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Analysis of the transgene structure in the BAC (ASS-EGFP) transgenic mice. (A) Overall view of BAC (ASS-EGFP) constructs showing relative positions of the EGFP transgene and the human ASS exons. Insertions of the EGFP gene with a polyadenylation signal into exon 3 (EGFP-pA) or an IRES-EGFP sequence into exon 16 of the ASS gene of BAC clone RP11-52B13 created the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) constructs, respectively. The lengths of the 5′ and 3′ human genomic sequences included in the BAC construct and the ASS structural gene are shown. Wavy line at the end represents the vector sequence. (B) Determination of transgene copy number. Genomic DNAs from offspring of the second generation transgenic mice (F2) was digested with EcoRI and Southern blot analysis was performed using an EGFP probe. The horizontal grey arrows are schematic representation of the transgene; the green bars represent the EGFP sequence; downward arrows indicate the EcoRI sites shown with expected sizes of the hybridized EcoRI fragments. The signal of a BAC (ASS-Ex3-EGFP) DNA preparation with predetermined copy number was used to estimate the transgene copy number in the mouse lines. The abbreviated transgenic line designations 3G, 3 J and 16E, 16H were two different lines each from the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) transgenes, respectively; the abbreviated designations are also used in other figures. (C) Confirmation of head-to-tail transgene integration. The PstI sites (downward arrows) and the expected 1,044-bp head-to-tail hybridized fragments are shown. The size of junction fragment may be greater than 939 bp (>939 bp) which is determined by position of the next PstI site in the mouse genome. (D) Monitoring of ASS-EGFP transgene transmission by fluorescence signals of tail samples. Pedigree analysis of the transgenic line Tg (ASS-Ex16-EGFP) 16 F is shown. Upper and lower rows were observed by dissecting microscopy under fluorescent light or white light, respectively.
Mentions: To investigate transcriptional and post-transcriptional regulation of the ASS gene, we established a transgenic mouse system using a modified bacterial artificial chromosome (BAC) carrying the human ASS gene tagged with the enhanced green fluorescent protein (EGFP) reporter gene [20]. Two transgenic mouse lines were generated. One line was Tg (ASS-Ex3-EGFP) which carries the transcription reporter BAC (ASS-Ex3-EGFP) (Figure 1A), where EGFP was knocked-in at the initiation codon of the human ASS gene and EGFP transcription is terminated by a SV40 poly (A) signal. EGFP activities of Tg (ASS-Ex3-EGFP) mainly reflect promoter activities of the ASS gene. Another line Tg (ASS-Ex16-EGFP) carries the transcription/post-transcription couple reporter BAC (ASS-Ex16-EGFP) (Figure 1A), where EGFP with an internal ribosome entry site (IRES) is inserted into the terminal exon at site between the stop codon and the polyA signal of the ASS gene; in such a configuration, translation of EGFP in the bicistronic transcript is regulated by the IRES mechanism. EGFP activities thus expressed are subjected to both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. Using these transgenic mouse lines, we have taken liver, the organ for urea production, intestine and kidney that are responsible for arginine biosynthesis as a model for temporal and spatial expression analyses [20]. We found the expression of the EGFP reporter gene in the transgenic mice faithfully reproduced that of the endogenous gene, suggesting that sufficient ASS regulatory elements are included in the transgene. Moreover, comparison between the EGFP expression profiles of the two transgenic lines indicated the developmental and tissue-specific regulation is mainly controlled at the transcriptional level [20].Figure 1

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