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Understanding Transcriptional Enhanc...

Nicoletti, Sarah E.

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Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells./
作者:	Nicoletti, Sarah E.
面頁冊數:	107 p.
附註:	Source: Dissertation Abstracts International, Volume: 77-04(E), Section: B.
Contained By:	Dissertation Abstracts International77-04B(E).
標題:	Nanotechnology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3739217
ISBN:	9781339297613

Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells.
Nicoletti, Sarah E.

Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells. - 107 p.

Source: Dissertation Abstracts International, Volume: 77-04(E), Section: B.

Thesis (Ph.D.)--State University of New York at Albany, 2015.

With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement.

ISBN: 9781339297613Subjects--Topical Terms:

526235
Nanotechnology.

Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells.
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245 1 0 $a Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells.
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500 $a Source: Dissertation Abstracts International, Volume: 77-04(E), Section: B.
500 $a Adviser: Susan T. Sharfstein.
502 $a Thesis (Ph.D.)--State University of New York at Albany, 2015.
520 $a With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement.
520 $a Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies.
520 $a Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE.
520 $a Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further investigations into both epigenetic modifications and transcription factor interactions will help gain a better understanding of what characteristics make a higher producing CHO cell line. Discovery of these characteristics will aid in better genetic engineering strategies for future recombinant cell lines, improving productivity and shortening the clonal selection process.
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