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Analysis of the Effects of Spatial L...

Choi, Leo.

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Analysis of the Effects of Spatial Localisation of Transgenes on Expression of Recombinant Proteins in CHO-DG44 Cells.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Analysis of the Effects of Spatial Localisation of Transgenes on Expression of Recombinant Proteins in CHO-DG44 Cells./
Author:	Choi, Leo.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2012,
Description:	214 p.
Notes:	Source: Dissertation Abstracts International, Volume: 74-09C.
Contained By:	Dissertation Abstracts International74-09C.
Subject:	Bioengineering. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10029556

Analysis of the Effects of Spatial Localisation of Transgenes on Expression of Recombinant Proteins in CHO-DG44 Cells.
Choi, Leo.

Analysis of the Effects of Spatial Localisation of Transgenes on Expression of Recombinant Proteins in CHO-DG44 Cells. - Ann Arbor : ProQuest Dissertations & Theses, 2012 - 214 p.

Source: Dissertation Abstracts International, Volume: 74-09C.

Thesis (Ph.D.)--The University of Manchester (United Kingdom), 2012.

Chinese hamster ovary cell lines are commonly used as host for production of recombinant protein both in research and in the biotech industry. Recombinant cell lines are generated through random integration of multi-cistronic plasmid vector containing the genes of interest and selection marker gene into the host genome. The recombinant cell lines require several rounds of limiting clonal dilution to isolate stable high expressing clones. Stable high expression of the genes of interest is a rare and desired trait in recombinant clonal cell lines. Despite being clonal, cell lines eventually become heterogeneous and lose productivity.The Mammalian genome resides as packaged chromatin in the nucleus, which is a highly organized structure with specialized spatial and functional compartments. Increasingly, evidence is pointing to the interaction between plasmid and nuclear architecture as a factor that affects expression and stability. This project is based on the hypothesis that certain locations within the three-dimensional structure of the nucleus are more favourable for stable high-level expression of recombinant genes, integration of transgene into these specific location will produce high expressing stable cell linesTo prove the hypothesis a set of far-red reporter gene expressing recombinant clonal cell lines were generated to use as model cell lines. These cell lines were characterized over 80 days of continuous culture to determine their expression and stability. Flow cytometry results showed that all cell lines showed heterogeneity and gradual loss of expression of the far-red gene. Majority of the cell lines loss 80% of their gene copy number by day 25 of continuous culture.The possibility of using telomeric repeat sequences and nuclear bodies as nuclear landmarks was explored in order to develop tools that can be used to study the localization and interactions of the integrated recombinant genes and the CHO genome in the nuclear environment. Telomeric repeat sequences were far too numerous and scattered to use as nuclear landmark. While PML nuclear bodies and Cajal bodies appeared to be randomly positioned.The possibility of cross-species chromosome painting using FACS sorted Chinese hamster chromosome as paints was investigated, results showed that two copies of chromosome 1 and a copy of chromosome 4 along with segments of other chromosomes 7 and X&Y are conserved in the CHO genome. This finding allowed us to further explore the localization of chromosomes in the interphase nuclei. Results showed that the CHO cell equivalent of hamster chromosome 4 does not have a preferred radial localization in the interphase nuclei. FISH data also showed that the site of reporter gene integration is variable and that the integrated plasmid does not have a preferred radial position within the nuclei. These findings indicated that the CHO nuclei in our cell lines do not have a common nuclear organization.Subjects--Topical Terms:

657580
Bioengineering.

Analysis of the Effects of Spatial Localisation of Transgenes on Expression of Recombinant Proteins in CHO-DG44 Cells.
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