Generally CHO (DHFR -ve) cells on transfection with plasmid bearing DHFR gene + Gene of Interest and upon addition of MTX only thoose cells which take up plasmid (containing DHFR + Gene of Interest ) will survive others will die.
My doubt 1 is : Generally DHFR is involved in De novo synthesis of Nucleotides, then how the nucleotides are synthesized in CHO (DHFR -ve ) cells?
My doubt 2 is : CHO (DHFR -ve ) cells lack DHFR so they couldn't use De novo pathway for nucleotide syntheis but they can use salvage pathway, then after transfection with Plasmid (containing DHFR + Gene of Interest) all the cells will survive due to operation of salvage pathway, now how to distinguish between the transfected cells vs Un transfected cells.
I'm confused with this DHFR-MTX selection system, could someone please help me to understand this concept, Also please share any referance material.
Hello Vikram Kumar
One of the ways in which maximum amount of protein can be achieved is by using gene amplification. Gene amplification is achieved by drug resistance. In this case (DHFR-MTX selection system) CHO cells are subjected to increasing concentration of MTX making them resistance to MTX via gene amplification.
In transfected cells the recombinant DNA consists of the GOI closely linked to the gene for DHFR. When resistance to MTX is achieved through gene amplification it causes overproduction of DHFR. So, only those CHO cells which are transfected will have increased copies of DHFR gene, and so higher levels of enzyme and therefore will survive in this DHFR-MTX selection system.
The untransfected CHO cells which are DHFR negative and since they lack the recombinant DNA consisting of the GOI closely linked to the gene for DHFR won’t be able to survive.
Also, in CHO (DHFR-ve) cells that lack DHFR cannot use De novo pathway for nucleotide synthesis, but they can use the Salvage pathway as you mentioned. The Salvage pathway involves the conversion of thymidine to thymidylate and occurs in the mitochondria and cytoplasm catalyzed by thymidine kinase 2 (TK2) and thymidine kinase 1 (TK1), respectively. However, Salvage pathway synthesis of dTTP is not sufficient to sustain mtDNA replication and therefore requires De novo dTMP synthesis, which involves the conversion of dUMP to dTMP, catalyzed by the tetrahydrofolate (THF)-dependent enzymes like serine hydroxy methyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase (DHFR).
So, the untransfected cells i.e. CHO (DHFR -ve) will not survive anyway in DHFR-MTX selection system.
Please refer to the research articles for more details
https://acsjournals.onlinelibrary.wiley.com/doi/pdf/10.1002/1097-0142(19860515)57:10%3C1912::AID-CNCR2820571004%3E3.0.CO;2-O
Article Identification of a de novo thymidylate biosynthesis pathway...
I hope this helps
Best,
Malcolm Nobre Thank you very much for your time and patiently replying clarifying my doubt.
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