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Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene.

Yong CS, Sharkey J, Duscio B, Venville B, Wei WZ, Jones RF, Slaney CY, Mir Arnau G, Papenfuss AT, Schröder J, Darcy PK, Kershaw MH - PLoS ONE (2015)

Bottom Line: Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2).Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer.This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Research Program, Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, 3010, Australia.

ABSTRACT
The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.

No MeSH data available.


Related in: MedlinePlus

Read alignments surrounding transgene integration.The screenshots from the IGV genome viewer show the integration site of the transgene on chromosome 5. A) displays the site and its surroundings. Grey reads are concordantly mapped reads. The colored reads indicate that the other half of the fragment maps discordantly. The teal colored reads are those that have mates on the Her2 transgene. Multi-colored segments at the ends of reads highlight soft-clipped portions of reads. There are three clusters of soft-clipped reads. Two around the integration site, and another block upstream that is unrelated to the integration. B) shows the integration in more detail. The proximity to exon 3 of the gene can be seen at the bottom. The right block of soft-clipped reads contains sequence that corresponds to just upstream of the transgene coordinate 4025 (the insert). The left block’s sequence derives from transgene coordinate 2972 and marks the return to chromosome 5 at the end of the concatemer.
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pone.0136817.g003: Read alignments surrounding transgene integration.The screenshots from the IGV genome viewer show the integration site of the transgene on chromosome 5. A) displays the site and its surroundings. Grey reads are concordantly mapped reads. The colored reads indicate that the other half of the fragment maps discordantly. The teal colored reads are those that have mates on the Her2 transgene. Multi-colored segments at the ends of reads highlight soft-clipped portions of reads. There are three clusters of soft-clipped reads. Two around the integration site, and another block upstream that is unrelated to the integration. B) shows the integration in more detail. The proximity to exon 3 of the gene can be seen at the bottom. The right block of soft-clipped reads contains sequence that corresponds to just upstream of the transgene coordinate 4025 (the insert). The left block’s sequence derives from transgene coordinate 2972 and marks the return to chromosome 5 at the end of the concatemer.

Mentions: The breakpoint detection performed with Socrates predicted 173 fusions in the data. To establish the insertion point we searched for overlap with the transgene sequence within the breakpoint set. There were three fusions that overlapped with the transgene: one from the transgene position 1 to the transgene position 6850, another from chromosome 5 at position 150719804 to the transgene (WAP-Her2) at position 4025, and finally from WAP-Her2 at position 2972 to chromosome 5 at position 150719794. A 10 nt duplication (150719804 to 150719794) was observed at these breakpoints in the host genome. The first fusion was caused by the transgene arranged in a 162 copy concatemer and therefore looping back onto its own start. The latter two breakpoints corresponded to the insertion site of the transgene into the native genome. Using whole genome sequencing, we were able to establish the WAP-Her2 transgene had inserted just 19 nucleotides upstream of exon 3 (150719823) in the Pds5b gene on chromosome 5 (Figs 3 and 4). The whole genome sequencing dataset can be found in the European Nucleotide Archive with accession number PRJEB9805.


Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene.

Yong CS, Sharkey J, Duscio B, Venville B, Wei WZ, Jones RF, Slaney CY, Mir Arnau G, Papenfuss AT, Schröder J, Darcy PK, Kershaw MH - PLoS ONE (2015)

Read alignments surrounding transgene integration.The screenshots from the IGV genome viewer show the integration site of the transgene on chromosome 5. A) displays the site and its surroundings. Grey reads are concordantly mapped reads. The colored reads indicate that the other half of the fragment maps discordantly. The teal colored reads are those that have mates on the Her2 transgene. Multi-colored segments at the ends of reads highlight soft-clipped portions of reads. There are three clusters of soft-clipped reads. Two around the integration site, and another block upstream that is unrelated to the integration. B) shows the integration in more detail. The proximity to exon 3 of the gene can be seen at the bottom. The right block of soft-clipped reads contains sequence that corresponds to just upstream of the transgene coordinate 4025 (the insert). The left block’s sequence derives from transgene coordinate 2972 and marks the return to chromosome 5 at the end of the concatemer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559457&req=5

pone.0136817.g003: Read alignments surrounding transgene integration.The screenshots from the IGV genome viewer show the integration site of the transgene on chromosome 5. A) displays the site and its surroundings. Grey reads are concordantly mapped reads. The colored reads indicate that the other half of the fragment maps discordantly. The teal colored reads are those that have mates on the Her2 transgene. Multi-colored segments at the ends of reads highlight soft-clipped portions of reads. There are three clusters of soft-clipped reads. Two around the integration site, and another block upstream that is unrelated to the integration. B) shows the integration in more detail. The proximity to exon 3 of the gene can be seen at the bottom. The right block of soft-clipped reads contains sequence that corresponds to just upstream of the transgene coordinate 4025 (the insert). The left block’s sequence derives from transgene coordinate 2972 and marks the return to chromosome 5 at the end of the concatemer.
Mentions: The breakpoint detection performed with Socrates predicted 173 fusions in the data. To establish the insertion point we searched for overlap with the transgene sequence within the breakpoint set. There were three fusions that overlapped with the transgene: one from the transgene position 1 to the transgene position 6850, another from chromosome 5 at position 150719804 to the transgene (WAP-Her2) at position 4025, and finally from WAP-Her2 at position 2972 to chromosome 5 at position 150719794. A 10 nt duplication (150719804 to 150719794) was observed at these breakpoints in the host genome. The first fusion was caused by the transgene arranged in a 162 copy concatemer and therefore looping back onto its own start. The latter two breakpoints corresponded to the insertion site of the transgene into the native genome. Using whole genome sequencing, we were able to establish the WAP-Her2 transgene had inserted just 19 nucleotides upstream of exon 3 (150719823) in the Pds5b gene on chromosome 5 (Figs 3 and 4). The whole genome sequencing dataset can be found in the European Nucleotide Archive with accession number PRJEB9805.

Bottom Line: Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2).Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer.This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Research Program, Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, 3010, Australia.

ABSTRACT
The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.

No MeSH data available.


Related in: MedlinePlus