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Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog.

Jaluria P, Betenbaugh M, Konstantopoulos K, Shiloach J - BMC Biotechnol. (2007)

Bottom Line: Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15.These results are consistent with previous studies on the functionalities of cdkl3 and cox15.For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Biotechnology Unit, Building 14A, Room 170, Bethesda, MD 20892, USA. pratikj@mail.nih.gov

ABSTRACT

Background: Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.

Results: Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15.

Conclusion: These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.

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Western blot analysis for several different cell lines indicating relative expression levels. These cells were derived from 3 different colonies that had already been screened and selected for expression of the transfected plasmid. At the time of analysis, these cells had been grown for over 6 days, post-transfection. The control cells were transfected with blank plasmids. 1 – HeLa 2 – HEK-293 3 – CHO
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Figure 3: Western blot analysis for several different cell lines indicating relative expression levels. These cells were derived from 3 different colonies that had already been screened and selected for expression of the transfected plasmid. At the time of analysis, these cells had been grown for over 6 days, post-transfection. The control cells were transfected with blank plasmids. 1 – HeLa 2 – HEK-293 3 – CHO

Mentions: To evaluate the impact of these two genes in cell culture, DNA plasmids containing either cdkl3 or cox15 were transfected into the attached HeLa cells. Cells transfected with only blank plasmids (i.e. containing neither cdkl3 nor cox15) served as the control. Each plasmid also contained the neomycin gene allowing cells to be selected based on resistance to geneticin. A number of clones were selected in this manner over the course of 1–2 weeks (post-transfection) and then assayed for expression of the translated protein using western blots. Figure 3 illustrates the protein expression of cells that have gone through this selection process. These blots indicate cells transfected with either cdkl3 or cox15 had significantly higher protein levels than the control cells. It should be noted that when the selection marker is not present in the culture media, these cell lines return to normal protein production levels within 5–6 passages.


Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog.

Jaluria P, Betenbaugh M, Konstantopoulos K, Shiloach J - BMC Biotechnol. (2007)

Western blot analysis for several different cell lines indicating relative expression levels. These cells were derived from 3 different colonies that had already been screened and selected for expression of the transfected plasmid. At the time of analysis, these cells had been grown for over 6 days, post-transfection. The control cells were transfected with blank plasmids. 1 – HeLa 2 – HEK-293 3 – CHO
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2164945&req=5

Figure 3: Western blot analysis for several different cell lines indicating relative expression levels. These cells were derived from 3 different colonies that had already been screened and selected for expression of the transfected plasmid. At the time of analysis, these cells had been grown for over 6 days, post-transfection. The control cells were transfected with blank plasmids. 1 – HeLa 2 – HEK-293 3 – CHO
Mentions: To evaluate the impact of these two genes in cell culture, DNA plasmids containing either cdkl3 or cox15 were transfected into the attached HeLa cells. Cells transfected with only blank plasmids (i.e. containing neither cdkl3 nor cox15) served as the control. Each plasmid also contained the neomycin gene allowing cells to be selected based on resistance to geneticin. A number of clones were selected in this manner over the course of 1–2 weeks (post-transfection) and then assayed for expression of the translated protein using western blots. Figure 3 illustrates the protein expression of cells that have gone through this selection process. These blots indicate cells transfected with either cdkl3 or cox15 had significantly higher protein levels than the control cells. It should be noted that when the selection marker is not present in the culture media, these cell lines return to normal protein production levels within 5–6 passages.

Bottom Line: Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15.These results are consistent with previous studies on the functionalities of cdkl3 and cox15.For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Biotechnology Unit, Building 14A, Room 170, Bethesda, MD 20892, USA. pratikj@mail.nih.gov

ABSTRACT

Background: Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.

Results: Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15.

Conclusion: These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.

Show MeSH