Genes can transfer into eukaryotic-cell genomes {transfection}|. Mice, plants, and yeast have only one transfection per thousand cells. DNA can go to cell nucleus but not enter genome, so gene expresses until DNA breaks down {transient expression}. Transfection takes time. Mammalian cell lines must be immortal. Cell culture requires many cells.
types
Inject DNA fragments into cell nucleus {microinjection}. Precipitate DNA fragments with calcium phosphate, so cell-culture cells absorb precipitated DNA by endocytosis. Make liposome lipid vesicles, with DNA inside, that can fuse with cell membranes and enter cells. Fire tungsten microbullets, with DNA fused to them, into plant cells, to penetrate cell wall.
types: virus
Viruses can transfect. Omitting coat proteins prevents virus formation, so cells do not die.
Monkey COS cells include most SV40-virus DNA and make T antigen, which binds to SV40 replication origin. Plasmids with SV40 replication origin can transfect COS cells. Vaccinia virus is large and can hold bacteriophage RNA polymerase. Plasmids with bacteriophage promoter can transfect cells and suppress cell mRNAs. Insect baculovirus DNA is large and can hold genes in coat-protein DNA.
types: retrovirus
Retroviruses can go into all mammalian cells. Retroviruses first place provirus DNA sequence in genomes and then make retroviral RNA. The next stage {packaging, virus} makes complete viruses by adding coat proteins. Then cells die and release viruses. For transfection, experimenters remove packaging genes from retrovirus {helper-free}, to prevent making complete viruses, so cells live.
Biological Sciences>Genetics>Recombinant DNA>Cloning>Transformation
4-Genetics-Recombinant DNA-Cloning-Transformation
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Date Modified: 2022.0224