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c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.

Stevanato L, Corteling RL, Stroemer P, Hope A, Heward J, Miljan EA, Sinden JD - BMC Neurosci (2009)

Bottom Line: After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks.In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain.The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

View Article: PubMed Central - HTML - PubMed

Affiliation: ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK. lara-stevanato@reneuron.com

ABSTRACT

Background: The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively.

Results: The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence.

Conclusion: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

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Alu sequence and c-mycERTAM assay development and validation. Gels showing the quality and purity of gDNA by agarose gel electrophoresis (A) and RNA by virtual gel produced by Agilent 2100 Bioanalyzer (B, RNA Integrity Number >9.4 as analyzed by Agilent RNA 600 nano kit [32]) isolated from the same sample. Standard curves used to determine: cell number by Alu sequence qPCR (C, Error 0.0300, efficiency 1.993; 3 replicates); absolute c-mycERTAM copy number by c-mycERTAM qRT-PCR (D, Error 0.0837 and efficiency 2.131; 3 replicates). All standard curves were generated from CTX0E03 gDNA diluted in rat gDNA or cDNA. Crossing point refers to the number of PCR cycles required to generate a detectable fluorescent signal generated on a Roche LC480 instrument. Positive control rat brain samples (B1 and B2) were grafted with approximately 300,000 CTX0E03 cells each and harvested immediately (E, F). Data shown is the total number of CTX0E03 cells in each tissue section as determined by Alu, where control is the number of viable cells in the cell suspension prior to injection as determined by counting using a haemocytometer (E); and total c-mycERTAM transcript copy number calculated per CTX0E03 cell detected in brain samples, where control is the number of copies per cell detected in vitro culture (F).
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Figure 1: Alu sequence and c-mycERTAM assay development and validation. Gels showing the quality and purity of gDNA by agarose gel electrophoresis (A) and RNA by virtual gel produced by Agilent 2100 Bioanalyzer (B, RNA Integrity Number >9.4 as analyzed by Agilent RNA 600 nano kit [32]) isolated from the same sample. Standard curves used to determine: cell number by Alu sequence qPCR (C, Error 0.0300, efficiency 1.993; 3 replicates); absolute c-mycERTAM copy number by c-mycERTAM qRT-PCR (D, Error 0.0837 and efficiency 2.131; 3 replicates). All standard curves were generated from CTX0E03 gDNA diluted in rat gDNA or cDNA. Crossing point refers to the number of PCR cycles required to generate a detectable fluorescent signal generated on a Roche LC480 instrument. Positive control rat brain samples (B1 and B2) were grafted with approximately 300,000 CTX0E03 cells each and harvested immediately (E, F). Data shown is the total number of CTX0E03 cells in each tissue section as determined by Alu, where control is the number of viable cells in the cell suspension prior to injection as determined by counting using a haemocytometer (E); and total c-mycERTAM transcript copy number calculated per CTX0E03 cell detected in brain samples, where control is the number of copies per cell detected in vitro culture (F).

Mentions: The analysis of the c-mycERTAM transcript expression per CTX0E03 cell, both in vivo and in vitro, was dependent upon the ability to sequentially purify genomic DNA (gDNA) and RNA from the same biological sample using the All DNA/RNA Kit (Qiagen). The gDNA preparation yielded one single band by agarose gel electrophoresis with no evidence of degradation or contaminating RNA (Figure 1A). The RNA preparation yielded the expected 28S and 18S bands, again with no degradation or DNA contamination visible (Figure 1B). Standard curves were then generated to determine: 1) the number of CTX0E03 cells present in a gDNA sample by Alu qPCR (Figure 1C); and 2) the number of c-mycERTAM transcripts present in a retro-transcribed RNA reaction sample (Figure 1D). The Alu sequence assay could reproducibly detect human CTX0E03 gDNA with as few as one CTX0E03 cell per reaction; whereas, the c-mycERTAM assay could detect as few as 10 copies per reaction.


c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.

Stevanato L, Corteling RL, Stroemer P, Hope A, Heward J, Miljan EA, Sinden JD - BMC Neurosci (2009)

Alu sequence and c-mycERTAM assay development and validation. Gels showing the quality and purity of gDNA by agarose gel electrophoresis (A) and RNA by virtual gel produced by Agilent 2100 Bioanalyzer (B, RNA Integrity Number >9.4 as analyzed by Agilent RNA 600 nano kit [32]) isolated from the same sample. Standard curves used to determine: cell number by Alu sequence qPCR (C, Error 0.0300, efficiency 1.993; 3 replicates); absolute c-mycERTAM copy number by c-mycERTAM qRT-PCR (D, Error 0.0837 and efficiency 2.131; 3 replicates). All standard curves were generated from CTX0E03 gDNA diluted in rat gDNA or cDNA. Crossing point refers to the number of PCR cycles required to generate a detectable fluorescent signal generated on a Roche LC480 instrument. Positive control rat brain samples (B1 and B2) were grafted with approximately 300,000 CTX0E03 cells each and harvested immediately (E, F). Data shown is the total number of CTX0E03 cells in each tissue section as determined by Alu, where control is the number of viable cells in the cell suspension prior to injection as determined by counting using a haemocytometer (E); and total c-mycERTAM transcript copy number calculated per CTX0E03 cell detected in brain samples, where control is the number of copies per cell detected in vitro culture (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2725042&req=5

Figure 1: Alu sequence and c-mycERTAM assay development and validation. Gels showing the quality and purity of gDNA by agarose gel electrophoresis (A) and RNA by virtual gel produced by Agilent 2100 Bioanalyzer (B, RNA Integrity Number >9.4 as analyzed by Agilent RNA 600 nano kit [32]) isolated from the same sample. Standard curves used to determine: cell number by Alu sequence qPCR (C, Error 0.0300, efficiency 1.993; 3 replicates); absolute c-mycERTAM copy number by c-mycERTAM qRT-PCR (D, Error 0.0837 and efficiency 2.131; 3 replicates). All standard curves were generated from CTX0E03 gDNA diluted in rat gDNA or cDNA. Crossing point refers to the number of PCR cycles required to generate a detectable fluorescent signal generated on a Roche LC480 instrument. Positive control rat brain samples (B1 and B2) were grafted with approximately 300,000 CTX0E03 cells each and harvested immediately (E, F). Data shown is the total number of CTX0E03 cells in each tissue section as determined by Alu, where control is the number of viable cells in the cell suspension prior to injection as determined by counting using a haemocytometer (E); and total c-mycERTAM transcript copy number calculated per CTX0E03 cell detected in brain samples, where control is the number of copies per cell detected in vitro culture (F).
Mentions: The analysis of the c-mycERTAM transcript expression per CTX0E03 cell, both in vivo and in vitro, was dependent upon the ability to sequentially purify genomic DNA (gDNA) and RNA from the same biological sample using the All DNA/RNA Kit (Qiagen). The gDNA preparation yielded one single band by agarose gel electrophoresis with no evidence of degradation or contaminating RNA (Figure 1A). The RNA preparation yielded the expected 28S and 18S bands, again with no degradation or DNA contamination visible (Figure 1B). Standard curves were then generated to determine: 1) the number of CTX0E03 cells present in a gDNA sample by Alu qPCR (Figure 1C); and 2) the number of c-mycERTAM transcripts present in a retro-transcribed RNA reaction sample (Figure 1D). The Alu sequence assay could reproducibly detect human CTX0E03 gDNA with as few as one CTX0E03 cell per reaction; whereas, the c-mycERTAM assay could detect as few as 10 copies per reaction.

Bottom Line: After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks.In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain.The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

View Article: PubMed Central - HTML - PubMed

Affiliation: ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK. lara-stevanato@reneuron.com

ABSTRACT

Background: The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively.

Results: The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence.

Conclusion: In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.

Show MeSH
Related in: MedlinePlus