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Engineering targeted chromosomal amplifications in human breast epithelial cells.

Springer S, Yi KH, Park J, Rajpurohit A, Price AJ, Lauring J - Breast Cancer Res. Treat. (2015)

Bottom Line: We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker.Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH.This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

View Article: PubMed Central - PubMed

Affiliation: The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, CRB 1 Room 146, 1650 Orleans Street, Baltimore, MD, 21287, USA.

ABSTRACT
Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

No MeSH data available.


Related in: MedlinePlus

AAV gene targeting strategy for engineering chromosomal amplifications. 5′ and 3′ homology arms (orange) flank a selection cassette containing an internal ribosome entry site (IRES, blue), the E. coli IMPDH gene (green), and a polyadenylation signal (PA, black). Targeting to the 3′ UTR of the gene of interest is selected for with mycophenolic acid (MPA) and identified by PCR screening. Subsequent selection with mizoribine leads to pressure to amplify the targeted cassette and flanking genes. Both exons of ZNF703 are depicted, but ERLIN2 is shown schematically as a single exon, and other genes are not shown
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Fig1: AAV gene targeting strategy for engineering chromosomal amplifications. 5′ and 3′ homology arms (orange) flank a selection cassette containing an internal ribosome entry site (IRES, blue), the E. coli IMPDH gene (green), and a polyadenylation signal (PA, black). Targeting to the 3′ UTR of the gene of interest is selected for with mycophenolic acid (MPA) and identified by PCR screening. Subsequent selection with mizoribine leads to pressure to amplify the targeted cassette and flanking genes. Both exons of ZNF703 are depicted, but ERLIN2 is shown schematically as a single exon, and other genes are not shown

Mentions: Model systems for gene amplification have generally relied on dominantly selectable enzymes such as DHFR and CAD [3, 17]. Cells expressing the enzyme are treated with an inhibitor, such as methotrexate or PALA, respectively, selecting for a subset of surviving cells with increased expression of the enzyme. In some cases, the increase in enzyme expression is caused by increased copy number of the locus encoding the enzyme. We reasoned that targeting such an amplifiable selectable marker to a genomic locus of our choice could lead to subsequent amplification of that locus and surrounding sequences under selective pressure from an inhibitor. We re-designed a recombinant adeno-associated virus (AAV) gene targeting vector for this purpose (Fig. 1; [12]). Since we did not wish to disrupt the coding sequence of our targeted gene, we designed homology arms targeting the selection cassette to the 3′ UTR of the chosen gene, downstream of the stop codon but upstream of the endogenous polyadenylation signal.Fig. 1


Engineering targeted chromosomal amplifications in human breast epithelial cells.

Springer S, Yi KH, Park J, Rajpurohit A, Price AJ, Lauring J - Breast Cancer Res. Treat. (2015)

AAV gene targeting strategy for engineering chromosomal amplifications. 5′ and 3′ homology arms (orange) flank a selection cassette containing an internal ribosome entry site (IRES, blue), the E. coli IMPDH gene (green), and a polyadenylation signal (PA, black). Targeting to the 3′ UTR of the gene of interest is selected for with mycophenolic acid (MPA) and identified by PCR screening. Subsequent selection with mizoribine leads to pressure to amplify the targeted cassette and flanking genes. Both exons of ZNF703 are depicted, but ERLIN2 is shown schematically as a single exon, and other genes are not shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4491111&req=5

Fig1: AAV gene targeting strategy for engineering chromosomal amplifications. 5′ and 3′ homology arms (orange) flank a selection cassette containing an internal ribosome entry site (IRES, blue), the E. coli IMPDH gene (green), and a polyadenylation signal (PA, black). Targeting to the 3′ UTR of the gene of interest is selected for with mycophenolic acid (MPA) and identified by PCR screening. Subsequent selection with mizoribine leads to pressure to amplify the targeted cassette and flanking genes. Both exons of ZNF703 are depicted, but ERLIN2 is shown schematically as a single exon, and other genes are not shown
Mentions: Model systems for gene amplification have generally relied on dominantly selectable enzymes such as DHFR and CAD [3, 17]. Cells expressing the enzyme are treated with an inhibitor, such as methotrexate or PALA, respectively, selecting for a subset of surviving cells with increased expression of the enzyme. In some cases, the increase in enzyme expression is caused by increased copy number of the locus encoding the enzyme. We reasoned that targeting such an amplifiable selectable marker to a genomic locus of our choice could lead to subsequent amplification of that locus and surrounding sequences under selective pressure from an inhibitor. We re-designed a recombinant adeno-associated virus (AAV) gene targeting vector for this purpose (Fig. 1; [12]). Since we did not wish to disrupt the coding sequence of our targeted gene, we designed homology arms targeting the selection cassette to the 3′ UTR of the chosen gene, downstream of the stop codon but upstream of the endogenous polyadenylation signal.Fig. 1

Bottom Line: We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker.Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH.This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

View Article: PubMed Central - PubMed

Affiliation: The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, CRB 1 Room 146, 1650 Orleans Street, Baltimore, MD, 21287, USA.

ABSTRACT
Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

No MeSH data available.


Related in: MedlinePlus