Knock out technology (final)

Gene knock out technology and animal models for human genetic

disorders

Submitted by: Dr. Vijayata

Gene knock out technology

• Knock outs can be produced by removing the gene or inducing

a mutation that disables its expression.

• The elimination of a single gene product from the genome can

yield important clues as to the function of that gene through

the phenotypic analysis of the resulting mutant.

Biotechnology 101, Science 101, ISSN 1931–3950 by Brian Robert Shmaefsky, First published in 2006 , GREENWOOD PRESS Westport, Connecticut London, pp138

Researchers who developed the technology for the creation of

knockout mice won Nobel Prize in the year 2007

• The Nobel Prize in Physiology or Medicine 2007 was awarded

jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver

Smithies "for their discoveries of principles for introducing

specific gene modifications in mice by the use of embryonic

stem cells".

Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414

The basic method for Gene knock out technology

• A targeting vector is created by flanking a mutated DNA

sequence (the gene of interest) with the DNA sequence

homologous to the endogenous gene.

• This vector is then introduced into mouse embryonic stem (ES)

cells where the mutant DNA replaces the native gene via

homologous recombination.

• The recombinant ES cells are then introduced into a fresh

blastocyst, where they mix with the cells of the inner cell mass.

Analysis of Genes and Genomes, Richard J. Reece, John Wiley & Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398

Transgenic and Gene-Knockout Rodents as Research tools for Cardiovascular Disorders, by Kapil Kapoor* & Madhu Dikshit, Scand. J. Lab. Anim. Sci. No. 2. 2005. Vol. 32

ES cells are harvested from the inner cell mass of a blastocyst and cultured in vitro. Here they can be genetically modified before being returned to a fresh blastocyst


• The blastocyst is then implanted into the uterus of a

pseudopregnant female and pups produced.

• Since the implanted blastocyst contains two different types of

ES cell (normal and recombinant), the resulting offspring will

be chimeric – some cells will contain the transgene, while

other will not.

• The chimeric pups are then crossed with wild type animals to

generate true heterozygotes, which can then subsequently be

inbred to create a homozygote.



• As the mutant gene encodes a major deletion or missense

mutation, mice homozygous for the targeted allele do not

express the native gene product and can be used to study the

effect of a total lack of a given protein.

• Breeding of various heterozygous and homozygous knockout

animals can be used to combine alterations in the expression

of multiple genes and to develop animal models of polygenic

diseases (Mauvais-Jarvis and Kahn, 2000).



Generation of Knockout Mouse

• Gene targeting by homologous recombination in embryonic

stem cells is a multi-step process.

• It begins with the generation of the targeting vector, which is

transferred by electroporation into the ES cells.

• The ES cells are cultured and analysed for the presence of the

homologously recombined DNA sequence; the targeted ES

cells are then injected into blastocyst stage embryos.


Embryonic Stem Cell Culture

• Embryonic stem (ES) cells are undifferentiated cells isolated

from the inner cell mass of a blastocyst (Evans and Kaufman,

1981).

• The crucially important factor about the progenitor cells of

these early embryos is that they are pluripotent – they have the

potential to differentiate into any cell type, including the germ

cells, of the subsequent embryo.


• ES cells in culture remain undifferentiated provided that they

are grown well separated from each other.

• It has been found that the presence of the cytokine leukaemia

inhibitory factor (LIF) is essential to ensure that ES cells do not

differentiate in vitro.

• For this reason, ES cells are generally grown on a feeder layer

of fibroblasts which secrete LIF into the culture medium.

• Most ES cells lines currently in use have been derived from the

129 strain of mouse which has an agouti coat colour genotype ;

this is useful when identifying chimeric mice.


ES cell colonies growing on a layer of fibroblast feeder cells. Healthy, undifferentiated ES cells.


Step 1. Generation of a Targeting Vector

• When designing and constructing a targeting vector, a number

of factors must be considered which will influence the type of

mutation to be introduced, the efficiency of targeting and the

ease with which successful targeting can be detected.

DNA homologous with the chromosomal/gene site of interest

• For successful and efficient targeting, the vector must contain

at least 5–10 kb of isogenic DNA homologous with the

sequence to be targeted.


• This homologous sequence is divided between the short arm of

homology (1–1.5 kb) and a long arm of homology (4–8 kb); this

permits easy screening of the ES clones.

• It is ideal to identify gene targeted colonies by PCR designed to span

the short arm of homology.

• It is known that the efficiency of homologous recombination is

decreased when there are base pair differences between the donor

and recipient DNA.

• For this reason, it is now common practice for the DNA used to

construct the targeting vector to originate from the same mouse

strain as the ES cells (i.e. isogenic DNA).Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414

Positive and negative selection cassettes

• Since gene targeting by homologous recombination occurs at

low frequencies (typically 105–106 of ES cells treated with

construct DNA) and the targeting construct is much more likely

to insert randomly into the genome, it is essential to be able to

screen ES cell colonies quickly and efficiently for successful

targeting.

• For this reason, most targeting vectors will be designed to insert

a positive selection cassette into the gene of interest.


• The most common positive selection marker is the neomycin

phosphotransferase (neor) gene, which when expressed in the

ES cell genome will render the cells resistant to treatment with

the antibiotic neomycin sulfate (G418).

• A negative selection marker, the HSV thymidine kinase (HSV-

tk) gene can also be used to enrich for gene targeted colonies.

Positive selection cassette Negative selection cassette


• The negative selection marker is cloned outside of the homologous

sequence in the targeting vector and will therefore not insert into

the genome when homologous recombination occurs.

• For example, the herpes simplex virus thymidine kinase gene

(HSVtk) when expressed in ES cells will produce a toxic product

in the presence of gancyclovir (a thymidine analogue), killing ES

cells expressing this gene.


Two homology arms flank a positive drug selection marker (neor). A negative selection marker (HSV-tk) is placed adjacent to one of the targeting arms. A unique restriction enzyme site is located between the vector backbone and the homology arm. When linearized for gene targeting, the vector backbone will then protect the HSV-tk from nucleases.

A schematic of a targeting vector:

Overview: Generation of Gene Knockout Mice, Bradford Hall1, Advait Limaye1, and Ashok B Kulkarni1,1 Curr Protoc Cell Biol. 2009 September ; CHAPTER: Unit–19.1217. doi:10.1002/0471143030.cb1912s44.

Targeting vector

Step 2. ES Cell Transfection

• The most efficient method for introducing the targeting vector

into the ES cells is by electroporation.

• The linearised vector DNA is electroporated into a large

number of ES cells in a single cell suspension; the cells are

then plated on to fresh feeder cells.

• Then, 24 h after electroporation, the selection process can

begin, which will kill cells which have not incorporated the

targeting vector by homologous recombination.


• The ES cells are cultured in media containing the drugs used for

selection for 7–10 days; this will enrich the population with

cells that have undergone homologous recombination;

however, it must be noted that this process is not 100% efficient.

Gene targeting by Homologous Recombination

• Homologous recombination is a DNA repair mechanism that is

employed in gene targeting to insert a designed mutation into

the homologous genetic locus.

• Targeted homologous recombination can be performed in

murine ES cells through electroporation of a targeting construct.


• The technique of gene targeting by homologous allows for the

introduction of engineered genetic mutations into a mouse at a

determined genomic locus. (generating mouse strains with

defined mutations in their genome)

• The most common application of gene targeting is to produce

knockout mice, where a drug resistance marker replaces an

essential coding region in a genetic locus.

Overview: Generation of Gene Knockout Mice, Bradford Hall1, Advait Limaye1, and Ashok B Kulkarni1,1Published in final edited form as: Curr Protoc Cell Biol. 2009 September ; CHAPTER: Unit–19.1217. doi:10.1002/0471143030.cb1912s44.


Targeting Vector

Genomic locus

Mutated locus

Homologous recombination results in the transfer of only the

neomycin resistance gene to the host cell.Analysis of Genes and Genomes, Richard J. Reece, John Wiley & Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398

Step 3. Identification of ES Cells Targeted by Homologous

Recombination

• To identify the ES cells that have undergone gene targeting by

homologous recombination, discrete colonies are identified

and picked.

• The colonies are dissociated into single cells by treatment with

trypsin, divided between two wells on duplicate microtitre

plates and cultured.


• The purpose of dividing the cells between duplicate plates is to

allow one plate of cells to be used to prepare DNA to identify

targeted ES cells and the cells from the second plate can be used

to inject into blastocysts.

• Genomic DNA is prepared from each ES cell clone, which is then

screened by PCR to identify clones in which homologous

recombination has occurred.

• Positive clones must then be further analysed, usually by Southern

blotting and DNA sequencing, to verify that all regions of the

targeting vector have undergone the desired recombination event.


Step 4. Injection of ES cells into Blastocysts

• Blastocysts, which are 3.5 day old embryos, are collected from

the uterus of the donor female.

• It is usual when using ES cells from the 129 strain of mouse to

collect blastocysts from a C57Bl/6 mother; this mouse line has

a black coat colour .

• ES cells carrying the desired mutation are treated to give a

single cell suspension.


• The ES cells are drawn up into the injection pipette by gentle

suction and the blastocyst to be injected is held by suction on

the holding pipette.

• The injection pipette is advanced into the cavity of the

blastocyst, which is known as the blastocoel, an 10–15 ES

cells are released .

• After injection, the embryos are cultured for a few hours to

allow them to re-expand slowly before being transferred to the

uterus of a pseudo-pregnant foster mother.

• Pups should be born 17 days later.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414

The blastocyst is held on the holding pipette by gentle suction (1).The injection needle containing ES cells is advanced into the blastocyst cavity (blastocoel) (2).where

the ES cells are released (3) and the injection needle is removed (4).

Injection of targeted ES cells into blastocysts


Step 5. Identification of Chimeric Mice and Breeding to Generate

Homozygous Mutant (Knockout) Mice

• Approximately 1 week after mouse pups are born, their coat colour

becomes apparent.

• At this stage, it is possible to identify agouti from non-agouti coat

colour.

• It is therefore possible to identify chimeric mice by their coat

colour if ES cells from the 129 mouse strain (agouti) have

contributed to the development of a C57Bl/6 embryo (non-agouti).


• Embryos in which the ES cells had made no contribution would

appear as wild-type C57Bl/6 (black), whereas those pups in

which the 129 ES cells had made a contribution would contain a

certain level of agouti coat colouring.

• Chimeric mice therefore contain some cells carrying the targeted

mutation on one allele and other cells which are wild type.

• To generate a gene knockout mouse, it is essential that some of

the germ cells carry the targeted mutation.


• To test for germline transmission of the mutation, chimeric mice

are bred to wild type mice; should germline transmission occur, a

proportion of the pups will be heterozygous for the targeted

mutation.

• Heterozygous mice can then be bred to produce mice homozygous

for the targeted mutation – gene knockout mice.


Generation of gene knockout mice by gene targeting in ES cells.


Generation of gene knockout mice by gene targeting in ES cells.

• The targeting vector is electroporated into the ES cells

• ES cells that have undergone homologous recombination are injected into

blastocyst stage embryos and these embryos are then transplanted to pseudo-

pregnant foster mothers.

• Chimeric offspring can be identified by their coat colour; these pups will carry

the targeted mutation carried by the injected ES cells.

• Chimeric offspring can then be mated to wild type mice to determine whether

they transmit the targeted mutation through the germline to give pups

heterozygous for the mutation.

• The heterozygous offspring can then be intercrossed to mice homozygous for

the mutation.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414

Advantages

• The integration site and therefore the gene modification are

highly specific.

• A variety of mutations can be achieved including null

mutations (gene knockout), deletion/rearrangement of large

regions of chromosomes, site-specific mutations.

• Recessive alleles can be studied.


Disadvantages

• Microinjection requires specialist, expensive equipment and

highly trained personnel.

• Process is very time consuming, taking 1.5–2 years to generate

a targeting vector, target ES cells, identify homologous

recombination events, microinject ES cells and test chimeric

pups for germline transmission of mutation.


• Process is expensive as it is labour intensive, requires expensive

equipment and the mouse husbandry costs will be high.

• Embryonic lethality – if the target gene is essential for

development of the embryo, then it will not be possible to study

the role of the gene in the adult mouse.


Animal models for human genetic disorders

• Many drugs, treatments and cures for human genetic diseases have

been developed with the use of animal models (Chakraborty et al.,

2009; Kari et al., 2007).

• When animal models are employed in the study of human disease,

they are frequently selected because of their similarity to humans in

terms of genetics, anatomy, and physiology.

• Also, animal models are frequently having advantage for

experimental disease research because of their infinite supply and

ease of handling (Simmons, 2008). Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

• Rodents are the most common type of mammal employed in

experimental studies.

• Among these rodents, the majority of genetic studies, especially

those involving disease, have employed mice, because their

genomes are so similar to that of humans.

• Mouse as an animal model provides a novel way to study a

signaling pathway in genetic disorder that is critical for embryonic

development (Barrott et al., 2011).

• Other common experimental organisms include fruit flies, zebra

fish, and chicks.Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

Rat

• The rat, being considerably larger than the mouse, has for

many years been the mammal of choice for physiological,

neurological, pharmacological, and biochemical analyses.

• The bigger size of rat is more advantageous than mouse for

collecting tissues (more tissue) and for surgeries.

• Rat models are also used for Human deafness diseases.

Vertebrate models

Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

• For example a hearing disorder due to mutation in Myosin XVA

gene causes DFNB3 phenotype in human (Irshad et al., 2012).

• The mouse and rat models used for this disease are shaker 2 mouse

and LEW/Ztm-ci2 rat respectively (Held et al., 2011).

• Genetic analysis in laboratory rats, however, is much less advanced

than in mice.

• It is partly because of the relatively high cost of rat breeding

programs and because until recently it has been much more difficult

to modify the rat germ line by gene targeting (Herrera and Ruiz,

2005). Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

Mouse

• The mouse (Mus musculus) is particularly well suited to genetic

studies and is an extensively used model of mammalian

development.

• Its short generation time like rat has allowed large scale

mutagenesis programs and extensive genetic crosses and various

features aid in mapping genes and phenotypes.

• Mouse are popular as an animal model because of their availability,

low cost, size, fast reproduction rate and ease of handling

(Simmons, 2008)Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

• The ability to construct mice with predetermined genetic modifications to

the germ line (by transgenic technology and gene targeting in embryonic

stem cells) has been a powerful tool in studying gene function and in

creating models of human disease (Davidson and Christiaen, 2006).

• These diseases include several types of cancer, heart disease,

hypertension, metabolic and hormonal disorders, obesity, diabetes,

osteoporosis, skin pigmentation diseases, deafness, blindness,

neurodegenerative disorders (such as Huntington's or Alzheimer's

disease), birth defects (such as cleft palate and anencephaly) and

psychiatric disturbances (including anxiety and depression) (Rosenthal

and Brown, 2007).


• Mouse models for a rare genetic disorder of the blood

platelets, May-Hegglin anomaly (MHA) showed same

symptoms as occur in humans (American Institute of Physics,

2011).

• Also in genetic prion disease, histopathological examination of

transgenic mice brain samples served as an ideal platform for

the investigation of this disease similarly to human (Levi et

al., 2011).


• Mouse models for deafness have revealed a variety of defective

structures and functions found in humans.

• In recent years, it has become essential to use mouse models as a

tool for studying genetic diseases, especially in cases of monogenic

disorders (Ganeshan et al., 2010).


Zebrafish

• There has been a very significant increase in the use of zebrafish

for the study of disease processes in humans.

• Zebrafish reproduce easily and quickly and have morphological

and physiological similarities to mammals.

• Zebrafish models have been developed for several human

diseases, including blood disorders, diabetes,

neurodegenerative diseases and muscular dystrophy

(Rubinstein, 2003).Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

Chick

• RE1-silencing transcription factor (REST) region in Human

phenotype DFNB55 for hearing impairment is also expressed in the

chicks.

• The REST gene was found to be expressed in supporting ear cells of

chick auditory epithelium (Irshad et al., 2005; Roberson et al.,

2002).


Frog

• Frogs of the genus Xenopus (African clawed frog) have been

particularly important models for investigating both

embryonic development and cell biology.

• There has also been seminal work on chromosome replication,

chromatin and nuclear assembly, cell cycle components and

cytoskeletal elements (Beck and Slack, 2001).


Invertebrate models

• Invertebrate models are often easy and inexpensive to

maintain, and can offer very large numbers of offspring and

rapid generation times.

• These characteristics make them ideally suited to high-

throughput genetic screening.

• The roundworm Caenorhabditis elegans and the fruit fly

Drosophila melanogaster are the two most widely studied

invertebrates (Segalat, 2007).Animal models for human genetic diseases ,Yasir Sharif and Saba Irshad* , Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

• D. melanogaster is employed in a wide variety of studies

ranging from early gene mapping, via linkage and

recombination studies to large scale mutant screens to identify

genes related to specific biological functions.

• Myo VIIa protein defect which causes usher syndrome in

human (Irshad et al., 2005) also lead to deafness in drosophila

(Todi et al., 2005).

• Caenorhabditis elegans is valuable for studying the

development of simple nervous systems and the aging process

(Spradling et al., 2006).


Thank You

Knock out technology (final)

Technology

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