Gene knock out technology uses embryonic stem cells to introduce targeted mutations into the mouse genome, allowing the study of gene function. A targeting vector containing the mutated gene sequence flanked by homologous DNA is introduced into embryonic stem cells. Cells with the mutation incorporated via homologous recombination are identified and injected into blastocysts, generating chimeric mice. Breeding of these mice can produce strains lacking the gene of interest, enabling investigation into the effects of its absence. This technology was developed in the 1980s-1990s and its creators were awarded the 2007 Nobel Prize in Physiology or Medicine.

1. Gene knock out technology and
animal models for human genetic
disorders
Submitted by:
Dr. Vijayata

2. 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

3. 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

4. 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

5. 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
Analysis of Genes
and Genomes,
Richard J. Reece,
John Wiley &
Sons, Ltd. 2004.
Chapter 13
Engineering
animals p 379 -
398

6. • 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.
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

7. • 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).
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

8. 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.
Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society

9. 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.
Analysis of Genes and Genomes, Richard J. Reece, John Wiley & Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398

10. • 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.
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

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

12. 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.
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

13. • 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

14. 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.
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

15. • 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
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

16. • 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.
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

17. 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.cb191
2s44.
Targeting
vector

18. 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.
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

19. • 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.
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

20. • 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.
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

21. 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

22. 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.
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

23. • 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.
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

24. 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.
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

25. • 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

26. 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
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

27. 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).
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

28. • 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.
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

29. • 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.
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

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

31. 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

32. 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.
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

33. 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.
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

34. • 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.
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

35. 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).

36. • 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

37. 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

38. • 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

39. 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

40. • 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).

41. • 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).
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

42. • 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).
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

43. 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

44. 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).
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

45. 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).
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

46. 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

47. • 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).
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

48. Thank You