Replicating nucleic acids can go into host organisms to make different organisms {transformation, DNA}|. Plasmids can enter by heat shock or electroporation. Bacteriophages can infect bacteria naturally. Transforming prokaryotic cells has high success rate.
Soy, maize, and other organisms {genetically modified organism} (GMO) can have deliberate genetic changes by genetic engineering.
Replacing genes with bad genes {knockout gene} makes animals that lack proteins. Transgenes can insert into normal gene positions, causing gene-function loss and affecting development.
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.
Changed or foreign genes can enter mouse embryo cells {transgenic mice} at chromosomal positions. Transgenic-mice descendants have changed or foreign genes and have new proteins.
organism
Mammals have cell and tissue interactions, so testing requires whole organisms.
process: injection
SV40, Moloney murine leukemia virus (MoMLV), or mouse mammary tumor virus (MMTV) microinjection can put changed or foreign DNA into cells. Cloned-gene microinjection into fertilized egg pronuclei can put changed or foreign DNA into cells.
process: cell addition
Mice embryos can change by adding altered cells. Mouse blastocysts have inner-cell {embryonic stem cell, blastocyst} (ES cell) layers, which can culture with fibroblasts or with leukemia inhibiting factor to prevent further differentiation. Embryonic stem cells can uptake and insert genes by homologous recombination. Then ES cells go into mouse embryos.
marker
Neo gene resists G418. ES cells with neo gene resist G418 and live.
embryonic development
In embryos, tissue-specific regulators express changed or foreign genes in one tissue but not different tissues. If changed or foreign genes are toxic, they destroy tissue {cell ablation, toxin}. Ablated cells prevent subsequent tissue development, allowing embryo location and function tracking {cell lineage study}. Retrovirus with E. coli lacZ reporter can trace tissue differentiation and cell migration.
Chinese-hamster ovary (CHO) cells {transgenic tissue} can track transgenic effects. Mammary glands can express transgenes.
4-Genetics-Recombinant DNA-Cloning
Outline of Knowledge Database Home Page
Description of Outline of Knowledge Database
Date Modified: 2022.0225