Genetic Engineering , Transgenic and Cloning

Genetic Engineering
Recombinant DNA Technology Collection of technique: purification, manipulation, use of genetic material Use of Enzyme DNA Copying ( replication) Conversion to RNA ( transcription) into protein (translation) > Manipulation DNA conversion into Protein; bacteria, yeast, viruses, transgenic animal, knock out mice, Diagnostic test, DNA based forensic

Transgenic

animal or other organism whose genetic composition has been altered by the addition of a foreign gene (the transgene)

 Use rDNA technology to introduce genes encoding desirable traits transfect gene into zygote implant zygote into surrogate mother screen offspring for ones with trait breed animals with trait to establish new genetic lines

Transgenic Animals Mice Cattle Sheep & Goats Pigs Birds Fish Cloning of Mammals

Transgenic Mice Applications - Why transgenic mice? model system for basic research gene regulation, cancer biology, development, etc. human genetic diseases model system for protein production (test in mice first, then try in cattle or other livestock)

Methodology
Retroviral Vector DNA Microinjection Engineered Embryonic Stem Cell

Retroviral Vectors for Generating Transgenic Mice

Vector
Integrates into mouse genome replication defective requires helper virus for replication

Disadvantages
infects only dividing cells size limit on insert ~8kbp could produce retroviruses (helper strain)

Transgenic Mice via Microinjection

Fertilized eggs collected from superovulated mouse Eggs injected with transgene directly into male pronucleus (larger than female pronucleus) Injected eggs reimplanted in surrogate mouse Screen pups for transgene Use transgenic mice for breeding

Embryonic Stem Cell Method for Transgenic Mice

Uses embryonic cells rather than eggs Transfection by retroviruses, liposomes, etc Transfected cells reimplanted into new blastocyst and allowed to develop

Embryonic Stem Cell Method for Transgenic Mice

Two main benefits
Integration of the DNA is to a specific site
Not a random integration

Can do Positive / Negative Selection

Negative selection of non-transfected cells Positive selection of cells with correctly integrated DNA

Testing for non-specific/nonhomologous integration

Some incorrect recombination events may escape negative selection if tk genes also eliminated Test all transgenic cells with PCR to see if transgenes are correctly integrated

Mouse Knockout Applications

Study of Gene Function mutation of genes overexpression of genes Model System Human genetic diseases knock out mouse gene homologous to human disease gene >250 mouse knockout models of human defects have been created

Transgenesis with High Capacity Vectors

Most transgenes derived from cDNAs
sometimes low expression somethimes need regulatory elements

Vectors for large inserts BACs, MACs, YACs etc. 100 to >1,000 kbp of DNA microinjection of artificial chromosomes
creation of transgenic mice making human antibodies ~1,000 kbp DNA segment containing human immunoglobulin locus

Transgenic Cattle - Applications

Increase the amount of k-casein for cheese production Reduced lactose milk Disease prevention Pharmaceutical protein production in milk mammary gland-specific promoter in urine bladder cell-specific promoter