scholarly journals Tight Binding of Plasmid DNA With Self-Assembled Tetramethylguanidinium Conjugated Polyethylenimine Suppresses Transfection Efficiency

2021 ◽  
Vol 3 ◽  
Author(s):  
Santosh Yadav ◽  
Ashwani Kumar Sharma ◽  
Pradeep Kumar
Keyword(s):  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Chinese Hamster Ovary Cells ◽  
Tight Binding ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Modified Polymers ◽  
Release Assay ◽  
Self Assembled ◽  
Linear Polyethylenimine

Here, we have demonstrated that on modification of linear polyethylenimine (lPEI, LP) with amphiphilic 3-bromopropyltetramethylguanidinium (PTMG) linker, the transfection efficiency exhibited by the modified polymers decreased while cell viability improved. A series of LP-PTMG polymers was synthesized by the reaction of varying amounts of 3-bromopropyl tetramethylguanidinium linker with lPEI (25 kDa). These modified polymers interacted efficiently with pDNA and formed nanosized complexes as shown by dynamic light scattering analysis. The size of the complexes in the series LP-PTMG/pDNA was observed in the range of ∼178–205 nm. The interaction of modified polymers with plasmid DNA was stronger than linear PEI as evidenced by heparin release assay which showed ∼83% pDNA release from LP-PTMG-3/pDNA complexes in comparison to ∼95% in lPEI/pDNA complexes on treatment with same amount of heparin suggesting the formation of self-assembled structures in modified polymers. The transfection studies in HeLa and Chinese hamster ovary cells showed a decrease in transfection efficiency of LP-PTMG polymers, the reason for this may be strong binding of modified polymers with pDNA due to accumulation of charge on the surface. This finding showed the significance of optimum binding of polymer and DNA to form polyplexes as well as release of DNA from the polyplexes.

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences

1983 ◽  
Vol 3 (4) ◽  
pp. 699-711
Author(s):  
R J Kaufman ◽  
P A Sharp ◽  
S A Latt
Keyword(s):  
Dihydrofolate Reductase ◽  
Plasmid Dna ◽  
Chinese Hamster Ovary Cells ◽  
Chromosome Rearrangement ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sequence Composition ◽  
Dicentric Chromosomes ◽  
Reductase Gene ◽  
Composition Structure

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.

scholarly journals Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

1983 ◽  
Vol 3 (4) ◽  
pp. 699-711 ◽  
Author(s):  
R J Kaufman ◽  
P A Sharp ◽  
S A Latt
Keyword(s):  
Dihydrofolate Reductase ◽  
Plasmid Dna ◽  
Chinese Hamster Ovary Cells ◽  
Chromosome Rearrangement ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sequence Composition ◽  
Dicentric Chromosomes ◽  
Reductase Gene ◽  
Composition Structure

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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