DNA fragments inserted into host nucleic acids can replicate in host organisms {cloning}|.
hosts: bacteria
Plasmids can insert up to 1000 bases. 50,000-base bacteriophage viruses can infect bacteria and can insert up to 15,000 bases. 300,000-base bacterial artificial chromosome DNA can have all bacterial-chromosome functional regions. Cosmids can hold 45,000 bases between cos sites. Gene-product secretion is preferable to harvesting cells.
In bacteria hosts, eukaryote proteins do not fold properly. Foreign proteins can kill bacteria. Bacteria have no post-translation enzymes.
hosts: yeast
Gene-product secretion is preferable to harvesting cells. In yeast hosts, proteases can destroy generated proteins.
For yeast hosts, replicating nucleic acid can be yeast artificial chromosomes.
Two-micron-circle yeast plasmid has replication origin that makes many copies per cell cycle. Other plasmids that use autonomously replicating sequence, sometimes helped by centromere sequence, make one or two copies per cell cycle. Yeast plasmids {shuttle vector} can work in bacteria.
Yeast vectors {integrating vector} with no replication origin integrate gene into yeast genome.
hosts: plants
For plant hosts, replicating nucleic acid can be Ti plasmid.
hosts: insects
For insect hosts, replicating nucleic acid can be baculovirus. Insect cell cultures have high costs. Gene-product secretion is preferable to harvesting cells.
hosts: mammals
For eukaryotic hosts, replicating nucleic acid can be virus or retrovirus. Mammalian cell culture has highest costs. Gene-product secretion is preferable to harvesting cells.
DNA fragment
DNA fragments can come from foreign organisms by cutting chromosomal DNA into DNA fragments using restriction enzymes. DNA fragments can come from mRNA by making cDNA from mRNA using reverse transcriptase and then making double-stranded DNA from cDNA. Synthesis methods can synthesize DNA.
polylinker
DNA fragments have polylinkers added at both ends, to allow nested cuts by different restriction enzymes.
insertion
DNA fragments can link into replicating nucleic acids using restriction enzymes to cut both nucleic acids and then allowing recombination.
selection
After replicating nucleic acids go into hosts, agents kill hosts if they do not have protecting genes in replicating nucleic acids. For example, bacteria with no plasmids die, because plasmids have genes to protect against antibiotics.
DNA
Host cells that live have DNA fragments, for extraction or secretion. Hybridization can test extracted or secreted DNA for DNA fragments. DNA sequencing can test for DNA fragments. Antibody binding or direct protein assays can test extractions or secretions for DNA-fragment gene products.
Toxic changed or foreign genes destroy tissue {cell ablation, cloning}.
Organisms, cells, and molecules can duplicate {clone}|.
Bacteria {colony, bacteria}| can grow on media.
DNA analyses can cleave chromosomes by restriction enzymes to make DNA fragments, separate fragments by size, and use fragment overlaps to mark relative restriction-enzyme-site positions {restriction map}.
Blunt ends can become sticky by attaching DNA {linker} containing recognition sites to blunt ends and then cleaving with restriction enzymes.
DNA fragments inserted into replicating nucleic acids can have many possible restriction enzyme sites {polylinker}, to allow nested cuts by different restriction enzymes.
Inheritable DNA-sequence positions {marker}| are restriction-enzyme cutting sites, fragment-length polymorphisms, genes, minisatellite DNAs, or microsatellite DNAs. Markers have inheritance patterns.
Replicated nucleic acids have added genes {marker gene}, to indicate foreign-DNA insertion and DNA replication.
bacteria
Hosts with added antibiotic resistance genes make proteins that resist antibiotics, whereas hosts with no such genes die. Beta-galactosidase gene makes protein that metabolizes galactose and makes color. Hosts with no beta-galactosidase gene have no color.
yeast
Yeast can grow without leucine if they have LEU gene, without histidine if they have HIS3 gene, without lysine if they have LYS2 gene, without tryptophan if they have TRP1 gene, and without uracil if they have URA3 gene.
plants
Genes {beta-glucuronidase gene} {GUS gene} can make protein that makes glucuronides. Plants have no glucuronides, so E. coli GUS genes can be markers for plants. Firefly luciferase gene makes light. Luciferase genes can be reporter genes for plants.
mammals
Thymidine kinase (tk) gene makes protein that makes thymidine triphosphate {thymidylate}. Mammalian cells (tk-) can have no thymidine kinase gene, so thymidine kinase genes can mark cells (tk+). Aminopterin inhibits all other thymidylate synthesis pathways, so only thymidine kinase gene can make thymidylate.
drugs
G418 inhibits protein synthesis and causes cell death. Aminoglycoside phosphotransferase (APH) gene makes protein that inactivates G418.
Methotrexate inhibits dihydrofolate reductase and causes cell death. Methotrexate-resistant dihydrofolate reductase (DHFR) gene makes protein that resists methotrexate.
Hygromycin-B inhibits protein synthesis and causes cell death. Hygromycin-B-phosphotransferase gene makes protein that alters hygromycin-B.
Mycophenolic acid inhibits GMP synthesis and causes cell death. Xanthine-guanine phosphoribosyltransferase (XGPRT) gene allows GMP synthesis from xanthine.
9-beta-D-xylofuranoyladenine (Xyl-A) damages DNA and causes cell death. Adenosine deaminase (ADA) gene metabolizes Xyl-A.
Replicating nucleic acids can have added genes {reporter gene} that catalyze reactions used to report that promoters are working or not, for gene-expression or transcription-factor studies. For example, chloramphenicol acetyltransferase gene (CAT) reacts with chloramphenicol. Reporter genes are after promoters, to provide direct promoter-activity assays.
If electric fields make holes in bacterial membranes {electroporation}, plasmids can enter bacteria.
Plasmids can enter bacteria during short high-heat periods {heat shock}|, in concentrated calcium-chloride solution.
Lipid vesicles {liposome}| with DNA or protein can fuse with cell membranes and enter cells.
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.
DNA or RNA sequences {cloning vector}| can contain DNA or RNA from other sources and can replicate in host organisms. Vectors include plasmids, phages, retroviruses, cosmids, baculoviruses, bacterial artificial chromosomes, and yeast artificial chromosomes.
Cauliflower-Mosaic-Virus promoters {35S promoter} can be in soy, maize, and other genetically modified organisms.
Vectors {Bacterial Artificial Chromosome} (BAC) derived from F-factor plasmids can clone 100,000-base to 300,000-base DNA fragments in Escherichia coli.
Cloning-vector plasmids {cosmid} can contain lambda-phage cos gene, infect E. coli, and clone DNA fragments up to 45,000 bases between phage-end cos sites.
Bacteria can have 5000-base circular DNAs {plasmid}| that can insert up to 1000 bases. Cells can have 10 to 200 independently replicating plasmids {relaxed-control plasmid}. Plasmids {stringent-control plasmid} can replicate together with bacterial chromosomes. Artificial plasmids can be cloning vectors.
For yeast hosts, replicating nucleic acids can be artificial DNA with all yeast-chromosome functional regions {yeast artificial chromosome} (YAC). YAC replicates like yeast chromosomes.
parts
Yeast artificial chromosomes contain autonomously replicating sequence, centromere (CEN), and telomeres.
gene size
YAC can hold 100,000 bases. Several YACs can undergo homologous recombination to create complete genes from fragments.
Yeast artificial chromosomes contain sequences {autonomously replicating sequence} (ARS) for replication.
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Date Modified: 2022.0225