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Transgenic Animals, Transgenic Animals, Knock Knock-Out Mice and Out Mice and Dolly Dolly

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Page 1: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Transgenic Animals,Transgenic Animals,KnockKnock--Out Mice andOut Mice and

DollyDolly

Page 2: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Transgenic Animals: A Focuson Transgenic Mice Studies

http://www.hku.hk/biochem/tgcentre/transcentre.html

Transgenic Animals: A Focuson Transgenic Mice Studies

http://www.hku.hk/biochem/tgcentre/transcentre.html

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Introduction Transgenic animals:

– Animals which have been genetically engineered tocontain one or more genes from an exogenous source.

– Transgenes are integrated into the genome.

– Transgenes can be transmitted through the germline toprogeny.

– First transgenic animal produced = “Founder Animal”

Transgenic animals:

– Animals which have been genetically engineered tocontain one or more genes from an exogenous source.

– Transgenes are integrated into the genome.

– Transgenes can be transmitted through the germline toprogeny.

– First transgenic animal produced = “Founder Animal”

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Introduction of foreign genesinto intact organisms

Procedure is basically the same regardless ofwhich animal is involved.

Integration usually occurs prior to DNAreplication in the fertilized oocyte.

– Majority of transgenic animals carry the gene in all oftheir cells, including the germ cells. Transmissionto next generation requires germline integration.

– Some integration events occur subsequent to DNAreplication giving rise to mosaic animals which may ormay not contain the transgene in its germline.

Procedure is basically the same regardless ofwhich animal is involved.

Integration usually occurs prior to DNAreplication in the fertilized oocyte.

– Majority of transgenic animals carry the gene in all oftheir cells, including the germ cells. Transmissionto next generation requires germline integration.

– Some integration events occur subsequent to DNAreplication giving rise to mosaic animals which may ormay not contain the transgene in its germline.

Page 5: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Procedure for ProducingTransgenic Mice

Three different breeding pairs of mice arerequired. Three different breeding pairs of mice are

required.

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First Breeding Pair:– Fertile male + superovulated female

• Fertile male = stud (changed regularly to ensureperformance)

• Superovulated female = immature female induced tosuperovulate

– Pregnant mare’s serum (=FSH) on day 1– Human Chorionic Gonadotropin (=LH) on day 3

• Mated on day 3• Fertilized oocytes microinjected on day 4 with foreign DNA

construct.• Microinjected oocytes are transferred to the oviducts of

surrogate mothers at end of day 4.

First Breeding Pair:– Fertile male + superovulated female

• Fertile male = stud (changed regularly to ensureperformance)

• Superovulated female = immature female induced tosuperovulate

– Pregnant mare’s serum (=FSH) on day 1– Human Chorionic Gonadotropin (=LH) on day 3

• Mated on day 3• Fertilized oocytes microinjected on day 4 with foreign DNA

construct.• Microinjected oocytes are transferred to the oviducts of

surrogate mothers at end of day 4.

Page 7: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Second breeding pair:– Sterile male + surrogate mother

• Sterile male produced through vasectomy• Surrogate mother must mate to be suitable recipient of

injected eggs• Mated on day 3• Microinjected oocytes from first breeding pair are

transferred to oviducts on day 4• Embryos implant in uterine wall and are born 19 days later.• Southern blotting techniques confirm presence and copy

number of transgenes.

Second breeding pair:– Sterile male + surrogate mother

• Sterile male produced through vasectomy• Surrogate mother must mate to be suitable recipient of

injected eggs• Mated on day 3• Microinjected oocytes from first breeding pair are

transferred to oviducts on day 4• Embryos implant in uterine wall and are born 19 days later.• Southern blotting techniques confirm presence and copy

number of transgenes.

Page 8: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

http://www.itba.mi.cnr.it/human_g...transgenic.html

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Third breeding pair:– Foster parents

• Fertile male + female mated to give birth on sameday surrogate mother

• Serves as foster parent if caesarian section isrequired for surrogate mother

Third breeding pair:– Foster parents

• Fertile male + female mated to give birth on sameday surrogate mother

• Serves as foster parent if caesarian section isrequired for surrogate mother

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Manipulation ofManipulation ofFertilized OocytesFertilized Oocytes

Refer to transparenciesand slides presented in

class.

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Flow Cytometry for the Analysis of Transgenic Mice

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Integration of Transgene intoOne Chromosome

Normally the transgene inserts into onechromosome giving rise to a heterozygote.– 50% probability of passing transgene onto offspring.

Two heterozygous mice may be bred to obtain ahomozygous line that contains the transgene onboth chromosomes.– 100% probability of passing transgene onto offspring.

Most transgenes are stably transmitted for manygenerations without detectable rearrangement.

Normally the transgene inserts into onechromosome giving rise to a heterozygote.– 50% probability of passing transgene onto offspring.

Two heterozygous mice may be bred to obtain ahomozygous line that contains the transgene onboth chromosomes.– 100% probability of passing transgene onto offspring.

Most transgenes are stably transmitted for manygenerations without detectable rearrangement.

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Mechanisms of DNAIntegration

Linear molecules integrate more efficiently than circularmolecules (~5x)

Once in the oocyte, the linear molecules circularize. Usually all of the molecules that integrate are on the same

chromosome and at the same site. Multiple copies are usually arranged in a tandem, head-

to-tail array. The size of the DNA molecule (0.7 – 50Kb) is not an

important parameter. The concentration and purity of the injected DNA is

critical (1-3 µg/ml maximum).

Linear molecules integrate more efficiently than circularmolecules (~5x)

Once in the oocyte, the linear molecules circularize. Usually all of the molecules that integrate are on the same

chromosome and at the same site. Multiple copies are usually arranged in a tandem, head-

to-tail array. The size of the DNA molecule (0.7 – 50Kb) is not an

important parameter. The concentration and purity of the injected DNA is

critical (1-3 µg/ml maximum).

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Working Hypothesis of DNAIntegration

The ends of the injected linear DNA integrate at breaks that occurspontaneously in the chromosome.

Other injected molecules which have circularized probablyrecombine with each other and the integrated copies to generate atandem, head-to-tail array.

Recombination is probably favored because of high local DNAconcentration and special properties such as the absence of normalchromatin structure.

The number of chromosomal breaks is presumably limitingexplaining the low number of integration events and why differentDNA molecules are usually integrated at the same site.

The ends of the injected linear DNA integrate at breaks that occurspontaneously in the chromosome.

Other injected molecules which have circularized probablyrecombine with each other and the integrated copies to generate atandem, head-to-tail array.

Recombination is probably favored because of high local DNAconcentration and special properties such as the absence of normalchromatin structure.

The number of chromosomal breaks is presumably limitingexplaining the low number of integration events and why differentDNA molecules are usually integrated at the same site.

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Gene Expression inTransgenic Mice

In order to discriminate the products of theinjected gene from those of an endogenouscounterpart, the injected gene must be marked insome way.– Mini-genes where exons are deleted of cDNA where

introns are absent.– Modification by insertion/deletion/mutagenesis of a

few nucleotides (e.g. the gain or loss of a restrictionendonuclease site).

– Hybrid genes where foreign epitopes are expressed ontransgenic products.

In order to discriminate the products of theinjected gene from those of an endogenouscounterpart, the injected gene must be marked insome way.– Mini-genes where exons are deleted of cDNA where

introns are absent.– Modification by insertion/deletion/mutagenesis of a

few nucleotides (e.g. the gain or loss of a restrictionendonuclease site).

– Hybrid genes where foreign epitopes are expressed ontransgenic products.

Page 19: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Tissue-Specific GeneExpression

Generally, if a tissue-specific gene is expressed at all,then it is expressed appropriately, despite the fact that ithas integrated at a different chromosomal location.– Trans-acting proteins involved in establishing tissue-specific

expression are capable of finding their cognate sequences andactivation transcription at various chromosomal locations.

– Levels of expression vary between founder animals aschromosomal position can influence accessibility of thetransgenes to these trans-acting transcription factors.

– Some founders do not express the transgene at all owing tointegration into heterochromatin domains where DNA ismethylated heavily (silent).

Generally, if a tissue-specific gene is expressed at all,then it is expressed appropriately, despite the fact that ithas integrated at a different chromosomal location.– Trans-acting proteins involved in establishing tissue-specific

expression are capable of finding their cognate sequences andactivation transcription at various chromosomal locations.

– Levels of expression vary between founder animals aschromosomal position can influence accessibility of thetransgenes to these trans-acting transcription factors.

– Some founders do not express the transgene at all owing tointegration into heterochromatin domains where DNA ismethylated heavily (silent).

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Prokaryotic Sequences Must beRemoved for Optimal Expression

Prokaryotic sequences derived from the plasmid orbacteriophage vector used for replication of the transgenein bacteria can be inhibitory or “poisonous” for sometransgenes.

Therefore, the transgene fragment requires purificationfrom contaminating vector sequence prior tomicroinjection.

Prokaryotic sequences derived from the plasmid orbacteriophage vector used for replication of the transgenein bacteria can be inhibitory or “poisonous” for sometransgenes.

Therefore, the transgene fragment requires purificationfrom contaminating vector sequence prior tomicroinjection.

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Possible Reasons for Lack ofTransgene Expression

Integration in cis-acting silencer sequences (the negative counterpartof enhancer elements) might be sites for covalent modification ofDNA (e.g. methylation) which might initiate condensation into aninactive chromatin configuration, or they might phase nucleosomes inan inappropriate manner.

The inadvertent loss of certain regulatory sequences during theproduction of the constructs (e.g. topoisomerase-binding sites,nuclear matrix-attachment sites).

Use of cDNA rather than genomic DNA. (Introns thought tocontribute to stability of mRNA and may even contain enhancersequences essential for tissue-specific expression. Flanking DNAmay also contain regulatory sequences.)

Integration in cis-acting silencer sequences (the negative counterpartof enhancer elements) might be sites for covalent modification ofDNA (e.g. methylation) which might initiate condensation into aninactive chromatin configuration, or they might phase nucleosomes inan inappropriate manner.

The inadvertent loss of certain regulatory sequences during theproduction of the constructs (e.g. topoisomerase-binding sites,nuclear matrix-attachment sites).

Use of cDNA rather than genomic DNA. (Introns thought tocontribute to stability of mRNA and may even contain enhancersequences essential for tissue-specific expression. Flanking DNAmay also contain regulatory sequences.)

Page 22: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Examples of Studies UtilizingTransgenic Mice

The Oncomouse– c-myc oncogene + MMTV sequences breast cancer

– Int-2 oncogene + viral promoter prostate cancer

To obtain abnormal expression of genes to study theireffects– Rat growth hormone + cadmium-inducible metallothionein

promoter

– Transgenic mouse was much larger, but also sufferedcomplications with fertility and organ diseases

The Oncomouse– c-myc oncogene + MMTV sequences breast cancer

– Int-2 oncogene + viral promoter prostate cancer

To obtain abnormal expression of genes to study theireffects– Rat growth hormone + cadmium-inducible metallothionein

promoter

– Transgenic mouse was much larger, but also sufferedcomplications with fertility and organ diseases

Page 23: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

To study developmentally regulated genes http://www.ucihs.uci.edu/anatomy/calofpix1b.html

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More Examples of StudiesUtilizing Transgenic Mice

“Pharm” animals (transgenic livestock)

– Bioreactors whose cells have been engineered tosynthesize marketable proteins

– DNA constructs contain desired gene and appropriateregulatory sequences (tissue-specific promoters)

– More economical than producing desired proteins incell culture

“Pharm” animals (transgenic livestock)

– Bioreactors whose cells have been engineered tosynthesize marketable proteins

– DNA constructs contain desired gene and appropriateregulatory sequences (tissue-specific promoters)

– More economical than producing desired proteins incell culture

Page 25: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Examples of Bioreactors

Naked human Hb frompigs

Human lactoferrin incows’ milk

Alpha-1-antitrypsin insheep

HGH in mouse urine(uroplakin promoters)

Human antibodies in mice(H and L chain tgenicshybridomas)

CfTCR in goats

Tissue plasminogenactivator (TPA) in goats

Human antithrombin IIIin goats

Malaria antigens in goats(vaccine)

Alpha-glucosidase inrabbits (Pompe’s disease

Naked human Hb frompigs

Human lactoferrin incows’ milk

Alpha-1-antitrypsin insheep

HGH in mouse urine(uroplakin promoters)

Human antibodies in mice(H and L chain tgenicshybridomas)

CfTCR in goats

Tissue plasminogenactivator (TPA) in goats

Human antithrombin IIIin goats

Malaria antigens in goats(vaccine)

Alpha-glucosidase inrabbits (Pompe’s disease

Page 26: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

http://www.bio.umass.edu/biotech/body_biotech.html

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http://www.vet.upenn.edu/catgcr/intro.html

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Transgenic Pigs for the Productionof Organs for Transplantation

Pig organs are rejected acutely due to thepresence of human antibodies to pig antigens.

Once human antibodies are bound to pig organs,human complement is activated and triggers thecomplement cascade and organ destruction.

Transgenic pigs with complement inhibitors havebeen produced and are gaining feasibility as asource of xenogeneic organs for transplantation.

Pig organs are rejected acutely due to thepresence of human antibodies to pig antigens.

Once human antibodies are bound to pig organs,human complement is activated and triggers thecomplement cascade and organ destruction.

Transgenic pigs with complement inhibitors havebeen produced and are gaining feasibility as asource of xenogeneic organs for transplantation.

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The KnockoutThe KnockoutMouseMouse

Page 34: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

What is a Knockout Mouse?

A really good-looking mouse?

A mouse in which a very specificendogenous gene has been altered in such away that interferes with normal expression,i.e. it has been knocked out.

A really good-looking mouse?

A mouse in which a very specificendogenous gene has been altered in such away that interferes with normal expression,i.e. it has been knocked out.

Page 35: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Why Produce KO Mice?

To study effects of gene products,biochemical pathways, alternative(compensatory) pathways, anddevelopmental pathways

To recreate human diseases in animals toestablish models to test the beneficialeffects of drugs or gene therapy.

To study effects of gene products,biochemical pathways, alternative(compensatory) pathways, anddevelopmental pathways

To recreate human diseases in animals toestablish models to test the beneficialeffects of drugs or gene therapy.

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Procedure for Generatinga KO Mouse

Gene alteration in KO mice is targeted to very specificgenes.

DNA must integrate at precise positions in the genome.

Integration of the altered gene takes place in embryonicstem cells ex vivo.

Verification of exact location of integration occurs beforethe ESC is introduced into blastocysts to become part ofthe developing embryo.

Gene alteration in KO mice is targeted to very specificgenes.

DNA must integrate at precise positions in the genome.

Integration of the altered gene takes place in embryonicstem cells ex vivo.

Verification of exact location of integration occurs beforethe ESC is introduced into blastocysts to become part ofthe developing embryo.

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Pluripotent ES Cells

Pluripotent ES cells are undifferentiated early embryoniccells derived from the inner cell mass of mouseblastocysts.

In vitro ES cells must be grown on a feeder layer offibroblasts to prevent them from differentiating.

Introduction of the transgene is achieved byelectroporation of retroviral infection.

The transgene must integrate via recombination, notrandomly.

Cells transfected successfully can be identified prior toinjection into blastocysts.

Pluripotent ES cells are undifferentiated early embryoniccells derived from the inner cell mass of mouseblastocysts.

In vitro ES cells must be grown on a feeder layer offibroblasts to prevent them from differentiating.

Introduction of the transgene is achieved byelectroporation of retroviral infection.

The transgene must integrate via recombination, notrandomly.

Cells transfected successfully can be identified prior toinjection into blastocysts.

Page 38: Transgenic Animals, Knock-Out Mice and Dollystaff.unila.ac.id/.../files/2020/04/Transgenic-Animals-2.pdfTransgenic animals: – Animals which have been genetically engineered to contain

Specific Gene Targeting in ESCells

Gene targeting can be achieved using geneconstructs designed for homologousrecombination. This technique can be used toeither:– Knockout functional genes to study their contribution

to different developmental or disease processes (nullmutations)

• Genes encoding β2m, MHC class I and II. CD2, Ii, TCR, Ig,IL-4, IL-2, FcεR, TAP1/2, RAG-2,and many more (>100)!

– Replace a functional gene for a mutated/non-functional gene to restore wild type phenotype .

• Gene encoding HGPRT in mice deficient for HGPRT (calledLesch-Nhyan syndrome in humans).

Gene targeting can be achieved using geneconstructs designed for homologousrecombination. This technique can be used toeither:– Knockout functional genes to study their contribution

to different developmental or disease processes (nullmutations)

• Genes encoding β2m, MHC class I and II. CD2, Ii, TCR, Ig,IL-4, IL-2, FcεR, TAP1/2, RAG-2,and many more (>100)!

– Replace a functional gene for a mutated/non-functional gene to restore wild type phenotype .

• Gene encoding HGPRT in mice deficient for HGPRT (calledLesch-Nhyan syndrome in humans).

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DNA Constructs forRecombination

DNA vectors contain the gene of interest whichhas been interrupted with an antibiotic resistancegene (hygromycin resistance, or G418resistance).

To ensure targeted integration has occurred, theflanking DNA contains the thymidine kinasegene. If TK integrates (random insertion), thenthe transfected cells die when grown in selectivemedia (gancyclovir).

DNA vectors contain the gene of interest whichhas been interrupted with an antibiotic resistancegene (hygromycin resistance, or G418resistance).

To ensure targeted integration has occurred, theflanking DNA contains the thymidine kinasegene. If TK integrates (random insertion), thenthe transfected cells die when grown in selectivemedia (gancyclovir).

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Selection of Targeted ES Cells

Gancyclovir resistant and G418 resistant ES cellsgrow into small clumps on top of feeder cells. The colonies of cells can be “picked” off and

transferred to new wells (at 0.3 cells per wellseeding density) containing feeder cells. When sufficient numbers of cells are obtained,

they are:– Frozen for safe storage– Analyzed by Southern blotting or PCR to determine

nature of integration event– Microinjected into the blastocoel cavity of blastocysts.

Gancyclovir resistant and G418 resistant ES cellsgrow into small clumps on top of feeder cells. The colonies of cells can be “picked” off and

transferred to new wells (at 0.3 cells per wellseeding density) containing feeder cells. When sufficient numbers of cells are obtained,

they are:– Frozen for safe storage– Analyzed by Southern blotting or PCR to determine

nature of integration event– Microinjected into the blastocoel cavity of blastocysts.

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http://www.criver.com/products/genetic_testing/dxgmon2.html

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Dolly and theDolly and theAdvancement ofAdvancement ofAnimal CloningAnimal Cloning

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How Was Dolly Made?