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Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells
1CSIRO Division of Molecular Science, Sydney Laboratory, North Ryde, NSW 2113, Australia.
Abstract:
Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.
Insights
Gene silencing in Chinese hamster ovary (CHO) cells was achieved using 5-methyl deoxycytidine triphosphate, leading to a "dual inheritance" pattern of the adenine phosphoribosyl transferase (APRT) gene.
Area of Science:
- Molecular Biology
- Epigenetics
- Genetics
Background:
- Chinese hamster ovary (CHO) cells are widely used in biological research.
- The adenine phosphoribosyl transferase (APRT) gene plays a crucial role in purine metabolism.
Purpose of the Study:
- To investigate gene silencing mechanisms in CHO cells.
- To analyze the epigenetic modifications associated with gene silencing.
- To demonstrate the concept of
- dual inheritance
- at the APRT locus.
Main Methods:
- Electroporation of CHO cells with 5-methyl deoxycytidine triphosphate to induce gene silencing.
- Selection of silenced cells using 2, 6-diaminopurine.
- Reactivation of silenced genes using 5-aza-cytidine.
- Bisulphite treatment of genomic DNA followed by PCR, cloning, and sequencing to analyze DNA methylation patterns.
- Comparison of gene silencing frequency in CHO strains D422 and K1.
Main Results:
- Successful induction of APRT gene silencing in CHO cells.
- Identification of DNA methylation in the promoter region of silenced APRT genes, while active genes remained nonmethylated.
- Demonstration of gene reactivation upon treatment with 5-aza-cytidine.
- Observation of a lower silencing frequency in CHO strain K1 compared to strain D422.
- Evidence supporting "dual inheritance" of active and silenced gene copies.
Conclusions:
- DNA methylation is a key mechanism underlying gene silencing in CHO cells.
- The study provides a method to track the inheritance of active, mutant, or silenced gene copies.
- The findings highlight the potential for epigenetic modifications to influence gene inheritance patterns.