Rekayasa Genetik & Transgenic

8/13/2019 Rekayasa Genetik & Transgenic

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Genetic Engineering ,

Transgenic and Cloning


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Genetic Engineering

Recombinant DNA Technology

Collection of technique: purification, manipulation, use ofgenetic 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


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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


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Transgenic Animals

Mice

Cattle

Sheep & Goats

Pigs

Birds

Fish

Cloning of Mammals


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Transgenic Mice

Applications - Why transgenic mice?

model system for basic research

gene regulation, cancer biology, development, etc.

human genetic diseasesmodel system for protein production

(test in mice first, then try in cattle or other livestock)


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Methodology

Retroviral Vector

DNA Microinjection

Engineered Embryonic Stem Cell

Retroviral Vectors for Generating


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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)


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Transgenic Mice via


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Transgenic Mice via

Microinjection

Fertilized eggs collected fromsuperovulated mouse

Eggs injected with transgene directly into

male pronucleus (larger than femalepronucleus)

Injected eggs reimplanted in surrogate

mouse Screen pups for transgene

Use transgenic mice for breeding


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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


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Testing for non-specific/non-

homologous 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


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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


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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


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Transgenic Cattle - Applications

Increase the amount of k-casein for cheese

production

Reduced lactose milk

Disease preventionPharmaceutical protein production

in milk

mammary gland-specific promoter

in urine

bladder cell-specific promoter


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Rekayasa Genetik & Transgenic

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