Encyclopedia of Experiments: Immunology
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Transcript
Begin with a tube containing a cationic lipid-based transfection agent and add recombinant expression plasmids.
The plasmid carries a gene encoding metallopeptidase-targeting recombinant antibodies and green fluorescent protein or GFP under one promoter, while the antibiotic resistance gene is under another promoter.
Positively charged lipids interact with a negatively charged plasmid, forming a complex.
Transfer the complexes into a multi-well plate containing mammalian cells, enabling the plasmid to enter the cytoplasm.
The plasmid reaches the nucleus and gets transcribed into mRNAs for antibiotic resistance proteins and bicistronic mRNAs encoding recombinant antibodies and GFPs.
These mRNAs lead to the production of recombinant antibodies, GFPs, and antibiotic resistance proteins.
Post-transfection, replace the medium with a serum-enriched medium.
Introduce an antibiotic, selectively eliminating non-transfected cells while the antibiotic-resistant transfected cells survive.
Using the fluorescence-activated cell-sorting technique, collect the green fluorescent cells expressing recombinant antibodies.
Seed the diluted transfected cells in a serum-free medium and incubate to produce recombinant antibodies against metallopeptidase.
