transgenic animals ananunes

8/2/2019 Transgenic Animals AnaNunes

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GloFish

Tobacco plant with GFPNeurons

TRANSGENICS


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Historic background

Concepts to remember

Transgenic technology and transgenic animals

Applica>ons of transgenic animals

Construc>on of transgenic animals

Ethical concerns

Overview


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Observa>on of inherited characteris>cs or spontaneous muta>ons.

Selec3ve breeding was a common prac>ce among farmers for the enhancement of chosentraits, e.g., increased milk produc>on.

1970s: First chimeric mice were produced (Brinster, 1974). The cells of two different

embryos of different strains were combined together at an early stage of development

(eight cells) to form a single embryo that subsequently developed into a chimeric adult,

exhibi>ng characteris>cs of each strain.

1981: DNA microinjec3on, the first technique to prove successful in mammals, was first

applied to mice (Gordon and Ruddle) and then to rats, rabbits, sheep, pigs, birds, and fish.

The term transgenic was first used by J.W. Gordon and F.H. Ruddle (had rapid

development in the use of gene>cally engineered animals with an increasing number of

applica>ons for the technology).

1986: Retrovirusmediated transgenesis (Jaenisch, 1986)

Embryonic stem (ES) cellmediated gene transfer (Gossler et al., 1986)

Fast development and rou3nely used lab technique in research, nowadays.

Historical background


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Plant or animal DNA

Bacterial plasmid

Specific

restric>on

enzyme

Specificrestric>on

Enzyme

(same)

Donor DNA

Plasmid DNA

(vector)

Each plasmid

contains

a differentdonor DNA

fragment

S>cky ends

combine

(complementary)

Engineered plasmid

incorporates into bacteria

which reproduce and

clone the gene from

donor cell that was spliced

into plasmid

Recombinant DNA

technology


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Homologous Recombina3on

Resection 5 ends are cut away

Strand invasion at homologous sitesRad52

DNA synthesis

DNA breakProtein

complex

Applica3on: To replace one allele with an

engineered construct but not affect any

other locus in the genome

We must know the DNA sequence of

the gene we want to replace

During meiosis and mitosis when

homologous chromosomes align along

the metaphase plane, recombina>on

takes place within the homologous

sequences. Recombina>on may take

place anywhere within the flanking DNA

sequences and the exact loca>on isdetermined by the cells. The end result

is a new piece of DNA inserted into the

chromosome. The rest of the genome is

unaltered.


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What are transgenic animals?

In this talk, the term transgenic animal includes:

animals that carry foreign DNA randomly integrated into their

genomes

animals generated by homologous recombina>on, allowingthe researcher to control the loca3on of the inserted DNA

(include KOs endogenous gene has been specifically

inac>vated Kis gene of interest has been added to the

genome or a na>ve gene has been enhanced)

Gene>c manipula>on of an organism (animal or plant) which permits stableintegra3on and expression of exogenous DNA fragments into the genome of

the organism.

Transgenic technology


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Medical research: iden>fy the func>ons of specific factors in complex homeosta>c systemsthrough over or underexpression of a modified gene (the inserted transgene); models of

human disease processes

Toxicology: responsive test animals (detec>on of toxicants);

Developmental gene3cs;

Molecular Biology: the analysis of the regula>on of gene expression makes use of the

evalua>on of a specific gene>c change at the level of the whole animal;

Pharmaceu3cal Industry: targeted produc>on of pharmaceu>cal proteins, drug produc>on

and product efficacy tes>ng;

Biotechnology: producers of specific proteins; gene>cally engineered hormones to increase

milk yield, meat produc>on; gene>c engineering of livestock and in aquaculture affec>ngmodifica>on of animal physiology and/or anatomy; cloning procedures to reproduce

specific blood lines;

Developing animals specially created for use in xenogra[ing.

Applica3ons of transgenic animals


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Transgenesis:

Aim of gene transfer studies influences

design of construct

Transgene expression

Transgene promoter: inducible expression

Gene targe3ng:

Condi3onal gene modifica3ons: CreloxP

Singlegene knockouts and Knockins

Gene knockdown (RNAi)

Reporter genes (GFAP tagging)

2) Design the construct according to the strategy

Construc3on of transgenic organisms

1) Delivery of DNA

Transfec3on (chemical, liposome

mediated, electropora>on)

Transduc3on (highlevel transient

expression, longterm stable

expression, stable transforma>on)

Directtransfer (microinjec>on, par>cle

bombardment)

Nuclear transfer / Stem cell transfer

Other examples:

Pelement transforma3on;

Recombinant Ti plasmid


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Construc3on of transgenics: delivering DNA into cells

TRANSFECTION

Chemical transfec3on: calcium phosphate ordextran sulfate opens transient holes in a cells

membrane, adming replacement DNA

Liposome transfer: liposome carries a gene into a

soma>c cell where the delivered gene may

replace a normal one

Electropora3on: electrical current opens transient

holes in a cells membrane, adming replacement

DNA

DIRECTPHYSICAL TRANSFER

Microinjec3on: >ny needle injects DNA into a

cell lacking that DNA sequence

Par3cle bombardment: metal pellets (gold or

tungsten) coated with DNA are shot with

explosive force or air pressure into recipient

cells

TRANSDUCTION

Virus: human gene inserted into a herpes viruswhich infects a human cell, where it is expressed

Retrovirus: RNA virus carrying RNA version of

human gene infects a soma>c cell. The gene is

reversed transcribed to DNA and inserts into a

human chromosome. Here it may produce a

missing or abnormal protein

STEM CELL TRANSFER

Transfer of embryonic stem cells into a developing

embryo by microinjec>on into blastocysts. The

resultant transgenic animal possesses a propor>on of

cells descended from the ES cell linage.

NUCLEAR TRANSFER

Transfer of a soma3c cell nucleus into an

enucleated egg.The egg is s>mulated with a shock

and aer many mito>c divisions, this single cell

forms a blastocyst with almost iden>cal DNA to

the original organism.


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TRANSFECTION

(1) Chemical

Low cost

Cells with inserted

DNA can be selected

by ampicillin

Mammalian cells in culture


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TRANSFECTION

(2) Liposomemediated


Highly efficient

Liposome has a membrane

similar to the cell allowing it to fuse

with the recipient cell membrane,

releasing the DN.A


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TRANSFECTION

() Electropora3on

Controlled milisecond electrical pulses are applied to the needle electrode, which form

an electric field, opening holes in the cell membrane, allowing DNA to enter the cell



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Process whereby foreign DNA is stably

introduced into another cell via a viral

vector.

TRANSDUCTION

Construc3on of transgenics:

delivering DNA into cells


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Retrovirusmediated transfer



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DIRECTPHYSICAL TRANSFER

Microinjec3on Par3cle bombardment



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SOMATIC CELL TRANSFER/ Pronuclear transfer

The transgene can integrate immediately

(mouse is transgenic) but more common the

DNA to integrate aer one or two cell

divisions, in which case the resul>ng mouse is

a mosaic containing both transformed and

nontransformed cells.

Newborn mice resul>ng from development

of the implanted embryos are checked byPCR or Southern blo>ng or a test for

tansgene expression for the presence of the

desired DNA sequence.

They will be heterozygous for the desired gene (transgenic founder).



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No more than 10% of mice progeny will be transgenic. While the technique can be applied

to other mammals, the transgenic progeny is much lower (< 1%). This is partly due to the

difficulty in handling eggs, and partly due to the lower survival rates.

Efficiency of microinjec3on


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The Cell Chapter 3

ESTRANSFER



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Efficient transfec>on Posi>venega>ve selec>on Gene targe>ng or inser>on of the gene Characteriza>on before animal genera>on Time and cost intensive

ESTRANSFER



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Recombinant Ti plasmid integra3on in Plants

protoplast



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Pelement transforma3on Drosophila melanogaster(1)


Highly mobile DNA element, which

can transpose from an

extrachromosomal element into a

chromosome. Generally, this

procedure results in incorpora>on of

a single copy of the transgene into

the Drosophila genome. In contrast,transgenic mice carry mul>ple copies

of the transgene incorporated into

their chromosomes. In both

organisms, chromosomal inser3on is

highly variable.

Individuals carrying the transgeneare recognized by expression of a

marker gene (ex: eye color) that is

also present on the donor DNA.


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Pelement transforma3on Drosophila melanogaster(2)


Flies that develop from injected

embryos will carry some germ cells

that have incorporated the

transgene: some of the progeny will

carry the transgene in all soma>c

and germline cells, giving rise to

pure transgenic lines. Individuals

carrying the transgene are

recognized by expression of a

marker gene. Although the

transgenes in Drosophila and mice

insert in chromosomal sites different

from the posi>on of the

corresponding endogenous gene,they usually are expressed in the

right >ssue and at the right >me

during development.


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Transgenesis:

Aim of gene transfer studies influences

design of construct

Transgene expression

Transgene promoter: inducible expression

Gene targe3ng:

Condi3onal gene modifica3ons: CreloxP

Singlegene Knockouts and Knockins

Gene Knockdown (RNAi)

Reporter genes (GFAP tagging)

2) Design the construct according to the strategy

Construc3on of transgenic organisms

1) Delivery of DNA

Transfec3on (chemical, liposome

mediated, electropora>on)

Transduc3on (highlevel transient

expression, longterm stable

expression, stable transforma>on)

Directtransfer (microinjec>on, par>cle

bombardment)

Nuclear transfer / Stem cell transfer

Other examples:

Pelement transforma3on;

Recombinant Ti plasmid


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Aim of gene transfer studies influences construct design

Gainoffunc3on transgeneAdd new func3onsto recipient individual

genomic gene

cDNA sequence

Lossoffunc3on transgeneGene knockout (Total inac>va>on)Gene targe>ng (Par>al inac>va>on / changeoffunc>on)

CreloxP system (Condi>onal inac>va>on)

Product can disrupt/interfere with host gene expression

an>sense RNA

dsRNA

small interfering RNA (siRNA)

Reporter transgeneStudying promoter ac>vi>es (>ssue/developmental stage/

cell/type specific expression)

Construct/ transgene design


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Transgene expression Transgene promoter

Transgene expression is regulated by

sequences present in the expression

construc3on and also by factors intrinsic to

the host genome.

Important considera3ons in constructdesign/ gene transfer experiments:

Control of transgene expression

Structure of the promoter

Op>miza>on of transla>onal start site

Inclusion suitable pep>de targe>ng signal

The transgene promoter defines

spa3al and temporal

paern of expression

Maximum control over transgene

expression in both cell lines and

animals is provided by inducible

promoters (switched on and off by

controlling the supply of a par>cular

chemical ligand).

In transgenic animals, it is oen desirable to express the transgene in par>cular >ssues or at par>cular developmental stages

By linking the transgene to a

suitable cell or stagespecific

promoter, the desired expression

paern may be achieved.


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Inducible expression: An>bio>cinducible (Tet On Tet Off) expression system

In this expression system, induc>on occurs at the level oftranscrip3on

(SLOW response to induc3on).

Construct design: promoters

Cons>tu>ve expression

TetRepressor


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Protein (X) fused with the estrogen receptor (ER) is

generally inac3ve sequestered into a complex

with heat shock protein 90 (Hsp90).

Induc3on is fast: requires only the dissocia>on of a protein complex and not

transcrip>on followed by protein synthesis.

Inducible expression: Hormoneinducible expression system

Construct design: promoters


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loxP recogni3on sequence

Type I Topoisomerase from P1 bacteriophage

that catalyzes sitespecific recombina>on of

DNA between loxP sites

Specific 34 bp sequences consis>ng of an 8bp

core sequence, where recombina>on takes

place, and two flanking 13bp inverted repeats

confering orienta>on.

CreloxP system (1)

Cre recombinase

Construct design: condi3onal gene inac3va3on


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The outcome of a Crelox recombina3on is determined by the orienta3on and loca3on of flanking

loxP sites. (A) If the loxP sites are oriented in opposite direc3ons, Cre recombinase mediates the

inversion of the floxed segment. (B) If the loxP sites are located on different chromosomes (trans

arrangement), Cre recombinase mediates a chromosomal transloca>on. (C) If the loxP sites are

oriented in the same direc3on on a chromosome segment (cis arrangement), Cre recombinase

mediates a dele>on of the floxed segment


CreloxP system (2)


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Typically, Cre and loxP strains are developed separately and crossed to produce a Cre

lox strain (Nagy 2000). The majority of Cre and loxP strains being developed fall into one

of the following categories:

* Cre expressing strains: contain a transgene that expresses cre under the control of a

widespread (general) or >ssuespecific (condi>onal) promoter. They are used to produce

general or condi>onal knockouts respec>vely.

* Inducible Cre strains: contain a transgene that expresses a modified form of Cre

recombinase that is nonfunc>onal un>l an inducing agent (such as doxycycline,

tetracycline, RU486, or tamoxifen) is administered at a desired >me point in embryonic

development or adult life

* LoxPflanked (floxed) strains: contain loxP sites flanking (on each side of) a cri>cal

por>on of a target gene or genomic region of interest

* Cre reporter strains: contain loxP sites in combina>on with visible (fluorescent or

lacZ) marker proteins used to trace Cre recombina>on success and/or altera>ons in gene

expression.


CreloxP system ()


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Mahias Zepper (Curnen)


CreloxP system (4)


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Gene targe3ng

Process of disrup>ng or muta>ng a specific gene>c locus in embryonic stem (ES) cells, usually withthe inten>on of making knockout or knockin mice by injec>ng those ES cells into blastocysts.

The en>re gene targe>ng process consists of the following major steps.

1) Linearize and purify the targe>ng construct; introduce it into ES cells by electropora>on; grow

clones under posi>ve selec>on by an>bio>cs; pick several hundred resistant clones into 96well

plates; split clones into duplicate sets; freeze one set and isolate DNA from the other set.

2) Genotype all clones by Southern blot using a probe specific for one end of the inserted DNA.

3) Expand clones that have the correct genotype (heterozygous for the targeted allele), freeze back

mul>ple vials of each clone, and prepare about 50 micrograms of genomic DNA from each clone for a

second genotyping assay (Southern blot or PCR) to characterize the other end of the inserted DNA.

Clones that are posi>ve for both genotyping assays may then be used for injec>on into blastocysts tomake chimeric mice.

Construct design


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Gene knockout by homologous recombina3on can inac>vate genes

at a predetermined locus within an intact cell

Inser4on vector

method

Replacement vector

method

.

Gene targe3ng: gene knockout

tkconfers sensi3vity to ganciclovir

Construct design: gene targe3ng


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Some genes are cri>cal early in development and simple knockout experiments aregenerally not helpful because death ensues at the early embryonic stage (ex NMDA)

Gene targe3ng: Gene knockin by introduc>on of subtle muta>ons

Subtle muta3on is present on the firsttarge3ng construct;

Intrachromosomal recombina>on leads to the

elimina>on of the marker gene and vector

backbone.

Inser3on vectors Replacement vectors

A second targe3ng construct is used toreplace the muta>on introduced by the first.

The second construct incorporates a counter

selectable marker outside the homology

region to avoid random integra>on.



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Gene targe3ng: Knockdown

RNA interferenceLong doublestranded RNAs can be used to silence the

expression of target genes in a variety of organisms and cell

types.

Upon introduc>on, the long dsRNAs enter a cellular

pathway that is commonly referred to as the RNA

interference pathway. First, the dsRNAs get processed into

2025 nucleo>de small interfering RNAs by an RNase IIIlikeenzyme called Dicer (ini>a>on step). Then, the siRNAs

assemble into endoribonucleasecontaining complexes

known as RNAinduced silencing complexes (RISCs),

unwinding in the process.

The siRNA strands subsequently guide the RISCs to

complementary RNA molecules, where they cleave and

destroy the cognate RNA (effecter step). Cleavage of

cognate RNA takes place near the middle of the region

bound by the siRNA strand.


High toxicity of transfected cells;

Can have effect on other host proteins


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A) Construc>on of GFPtaggedprotein B) Transgenic mice with GFP fused to anepithelial protein

A) The recombinant gene encodes a fusion protein that contains GFP at its C terminus.

B) This mouse contains a GFPlabelled transgene expressed throughout the body; the en>re mouse becomes

fluorescent when illuminated with UV light as at the boom. The same mouse is shown in normal light at the top.

Gene>cs: From genes to Genomes, 2/e. ( McGrawHill Companies, 2004)

Gene targe3ng: GFP tagging to study protein localiza>on

Construct design use of reporter genes to study expression


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Week 1Prepare DNA

construct

Week 2

Microinjec3on

and implanta3on

Week 5

Founder pups born

Week 8

Founder animals

weaned and genotyped

Week 1

Transgenic

founders mated

Week 16

F1 pups born

Week 19

F1s genotyped

germline transmission

Week 2

Colony expansion

Timeline for development of a transgenic mouse line

Backcross to founder for 10 genera>ons to clean background

Week 18

Chimeras

Mated for

wildtype

Week 22

Kos iden3fied by

Coat color

Week 9

Genotype for

Homologous

recombina3on

Week 12

Transfer into

Pseudopregnant

female

Week 1

Prepare KO

construct

Week 4Transform

ES cells

Week 5

Apply

selec3on

Week 7Expand

Transformed

cells

Week 15Chimeric

Animals born

Week 21

F1 genera3on

born

Week 27Begin planned

breeding

Classic transgenic

ESderived transgenic


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Why the mouse?

Of the model organisms which may be gene3cally modified, the mouse is:

The closest to humansMammal

The most complexIntegra>on of systems (endocrine, immune, nervous, etc.)

Gene3c manipula3on is extremely versa3leGainofFunc>on (Transgenesis)

LossofFunc>on (knockout)

ChangeofFunc>on (knockin);

Temporally and spa>ally restricted (Condi>onal)

Why the mouse?


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Mouse model of Human disease


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There are several areas where differences between mice and humans

could be expected to result in divergent disease phenotypes for

muta>ons in orthologous genes:

Differences in biochemical pathways Differences in development pathways Absolute 3me Differences in gene3c background

The recent comple>on of the mouse and rat genome sequences has

iden>fied a number of human genes that do not appear to have

counterparts in rodents.

Human vs Mouse


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Thoughul ethical decisionmaking cannot be ignored

Ethical issues include ques3ons such as:

Should there be universal protocols for transgenesis? Should such protocols demand that only the most promising research be permied?Is human welfare the only considera>on? What about the welfare of other life forms? Should scien>sts focus on in vitro transgenic methods rather than, or before, usinglive animals to alleviate animal suffering?

Will transgenic animals radically change the direc>on of evolu>on, which may result indras>c consequences for nature and humans alike?

Should patents be allowed on transgenic animals, which may hamper the freeexchange of scien>fic research?

Ethical implica3ons


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Take home messages

Plan carefully your experiment according to the research ques3on

Take into account the 3me required to obtain all the necessary steps.

If planning experiments with animals, always keep the number of animals to aminimum

Be aware of the ethical implica3ons of your work

If possible, take a course on how to handle animals for research and learn with

more experienced people


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Siegel, G.; Agranoff, B.; Albers, R.; Fisher, S.; Uhler, M., Basic Neurochemistry: Molecular,

Cellular, and Medical Aspects (1999), Lippinco, Williams & Wilkins, Philadelphia

Strachan, T. and Read, A., Human Gene3cs (1999), Garland Science, New York and London

Hartwell L., Hood L. , Goldberg M., Reynolds A., Silver L., Veres R., Gene3cs: from genes to

genomes 2nd Edi3on (2004), McGraw Hill

Background literature

Bruce Alberts, Alexander Johnson, Julian Lewis, Mar>n Raff, Keith Roberts, and Peter Walter,

Molecular Biology of the Cell, 4th edi3on, (2002), Garland Science,New York

transgenic animals ananunes

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