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11 Keratin Transgenic and Knockout Mice Functional Analysis and Validation of Disease-Causing Mutations Preethi Vijayaraj, Goran Söhl, and Thomas M. Magin Summary The intermediate filament (IF) cytoskeleton of mammalian epithelia is generated from pairs of type I and type II keratins that are encoded by two large gene families, made up of 54 genes in humans and the mouse. These genes are expressed in a spatiotemporal and tissue-specific manner from the blastocyst stage onward. Since the discovery of keratin mutations leading to epidermolysis bullosa simplex, mutations in at least 18 keratin genes have been identified that result in keratinopathies of the epidermis and its appendages. Recently, noncanonical mutations in simple epithelial keratins were associated with pancreatic, liver, and intestinal disorders, demonstrating that keratins protect epithelia against mechanical and other forms of stress. In recent years, animal models provided novel insight and significantly improved understanding of IF function in tissue homeostasis and its role in disease. Pathological phenotypes detected in mutant mice generated so far range from embryonic lethality to tissue fragility to subtlety, which often depends on their genetic background. This range implies at least a partial influence of yet unidentified modifier genes on the phenotype after the ablation of the respective keratin. To date, nearly all available keratin mouse models were generated by taking advantage of conventional gene-targeting strategies. To reveal their cell type-specific functions and the mechanisms by which mutations lead to disease, it will be necessary to use conditional genetargeting strategies and the introduction of point-mutated gene copies. Furthermore, conditional strategies offer the possibility to overcome embryonic or neonatal lethality in some of the keratin-deficient mice. Key Words: Blastocysts injection; Cre/LoxP system; epidermolysis bullosa simplex (EBS); epidermolytic hyperkeratosis (EHK); pachyonychia congenita; embryonic stem (ES) cell culture; Flp/FRT system; genomic cluster knockout; intermediate filaments; knockout mice; tetraploids; transgenic mice. From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols Volume I, Emerging Strategies for Targets and Biomarker Discovery Edited by: M. Sioud © Humana Press Inc., Totowa, NJ 203
204 Vijayaraj, Söhl, and Magin 1. Introduction 1.1. Characterization of Genetically Altered Keratin Mice 1.1.1. Two Families of Keratin Genes Of the currently known and characterized intermediate filament (IF) genes (1), keratin genes establish the most complex and conserved gene family in mammals. They are encoded by a total of 54 members in the human and mouse genome, located in two different arrays and designated as type I and type II clusters (2,3) (see Fig. 1). All type I keratin genes, except for K18, are clustered on human chromosome 17q21 in synteny to mouse chromosome 11D, whereas the type II cluster, localized on human chromosome 12q13, is in synteny to mouse chromosome 15F (4) (The International Human Genome Sequencing Consortium). The human keratin I gene cluster comprises totally 977 kilobases (kb), consists of 27 functional and 4 pseudokeratin type I genes as well as high and ultrahigh sulfur keratin-associated proteins (KPs) (5). In the mouse, the keratin I gene cluster is approx 1,034 kb and highly orthologous in terms of number as well as orientation of the orthologous keratin genes and KPs (3). The central KP subdomain (362 kb in human and 466 kb in mouse) divides this cluster into a centromeric and telomeric portion and contains in human 29 genes coding for high and ultrahigh sulfur (depending on their cysteine content) hair KPs, being transcribed in the upper cortex of the hair shaft (5). This KP subdomain is surrounded by keratin genes of the inner root sheath of hair follicles (6) and several hair keratin genes (3). Thus, the human and mouse keratin I gene cluster can be considered as highly organized in subdomains containing cytokeratins (K9–K24), hair keratins (K25–K36), and inner root sheath keratins (K38–K41) (Fig. 1). The human keratin II gene cluster is located on chromosome 12q13.13, comprises 783 kb and contains 26 functional type II genes, 5 type II pseudogenes, and 3 keratin unrelated pseudogenes. In the mouse type II cluster, however, there are 27 functional type II genes, mostly arranged in the same orientation. Interestingly, both type II clusters (mouse and human) end up with a type I keratin gene, the K18 gene. However, the type II clusters of both species diverge more than their type I clusters, which could be considered as highly identical. The human type II cluster contains shortly upstream of the K18 gene three nonkeratin genes as well as the cornea-specific keratin gene K3, all of which lack a mouse ortholog. This difference, most likely, resulted from gene duplication in human and other species (7) or vice versa from a K3 gene lost in the mouse genome. 1.1.2. Keratin Assembly, Structure, and Modifications Similar to other intermediate filament protein, the secondary structure of keratin proteins can be subdivided into three subdomains: a central, largely α-helical
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 281 Fig.
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Overview of Immune System 283 then
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Overview of Immune System 285 expre
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Overview of Immune System 289 solub
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Overview of Immune System 293 (unre
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Overview of Immune System 297 the c
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Overview of Immune System 299 (44).
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Overview of Immune System 301 demon
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Overview of Immune System 307 some
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Overview of Immune System 309 sera
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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