Knock out technology (final)

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 of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414

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

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 Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

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 Journals

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 Journals

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 Punjab, Lahore, Pakistan. . African Journal of Biotechnology Vol. 11(86), pp. 15200-15205, 25 October, 2012, ISSN 1684-5315 ©2012 Academic Journals

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). 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

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