Mouse Models in Preclinical Studies for Pachyonychia Congenita

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1 Mouse Models in Preclinical Studies for Pachyonychia Congenita
Jiang Chen, Dennis R. Roop  Journal of Investigative Dermatology Symposium Proceedings  Volume 10, Issue 1, Pages (October 2005) DOI: /j x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Generation of transgenic and knock-in/knockout mouse models. (A) Generation of transgenic mouse model. Recombinant DNA carrying genetic information is engineered before it is injected in the male pronucleus of a fertilized oocyte. The recombinant DNA is inserted randomly into the genome of the oocyte. The oocyte develops into an early mouse embryo and the embryo is implanted into the uterus of a pseudopregnant mouse. The mouse that develops from the modified oocyte carries the recombinant transgene in a random location, therefore, transgene expression may occur in an uncontrolled fashion. (B) Generation of knock-in or knockout mouse model. Defined genetic modification is constructed in a targeting vector, which carries a drug resistant selection marker and homologous regions. The homologous regions define the specific region of genomic integration. The targeting vector is electroporated into mouse embryonic stem (ES) cells. Correctively targeted ES cells are selected by antibiotics and expanded. ES cells are injected into an early mouse embryo and the embryo is implanted into the uterus of a pseudopregnant mouse. Chimeric mice, which develop from the mixture of modified ES cells and cells of the host embryo, have fur of both colors. These chimeric mice are mated with normal mice to obtain mice that are derived exclusively from the modified ES cells. Germline transmitted mice carry the designated genetic change (modification of a gene for knockin or deletion of a gene for knockout). The change occurs at the genomic locus of the gene of interest, therefore, its gene expression (for knock-in) is under the same control as for the endogenous wild-type allele. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Schematic diagram of the K14-CrePR1 inducible excision system. K14 promoter directs expression of the fusion protein CrePR1 in the basal keratinocytes, and it remains inactive in the cytoplasm. Upon induction with RU486, the fusion protein translocates into the nucleus, where Cre excises the neo-cassette flanked by two loxP sites. *R154C mutation. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Focal induction of blisters in the inducible epidermolytic hyperkeratosis (EHK) mouse model. The left panel shows blister formation on the forelimbs and chest 4 d after treatment. Unlike epidermolysis bullosa simplex (EBS)-induced blisters, EHK blisters persist after treatment with the activator is stopped. Shown in the right panel is a mouse at 3 mo following blister induction. But, persistent blistering has now been observed for over 1 y. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Focal induction of blisters in the inducible epidermolysis bullosa simplex (EBS) mouse model. The left panel shows the induction of a blister 2 d following treatment with the activator on one paw. Ten days following blister induction (middle panel). Six months after blister formation the paw appears normal and no additional blisters develop (right panel). Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Upon topical application of an inducer to the skin, the mutant alleles are activated by excision of the neo-cassettes. In the epidermolysis bullosa simplex (EBS) model, the mutant K14 allele is not only activated in epidermal stem cells, but also expressed in these cells. Therefore, these cells are fragile and are replaced by non-phenotypic stem cells (mtK14neo) migrating in from the surrounding non-treated area. Although the neo-cassette is excised from the mutant K10 allele in stem cells, the gene is not expressed in stem cells, but only in the differentiated progeny of these cells in the suprabasal layers of the epidermis. Consequently, there is no selective pressure against stem cells containing the mtK10loxP allele. These stem cells persist and give rise to islands of mutant cells that result in persistent lesions for the life of the mouse. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 The “humanized” MK6a N160K targeting vector. (A) MK6a genomic locus and the backbone of the targeting vector. The figure illustrates that all exons and a large fragment of the 5′ untranslated region (UTR) is cloned in a TK vector. (B) The linearized targeting vector. Part of the MK6a sequence in exon 1 was replaced by a fragment of mutant (*) HK6a (yellow box) sequence. A neo-cassette is inserted in intron 1. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Strategy to generate inducible transgenic mice expressing the N160K mutation. (A) Genomic locus of MK6a. Exons are represented by black boxes. (B) Predicted structure of the “humanized” mutant MK6a locus. The neo-cassette with multiple polyA signals in intron 1 prevent gene transcription from the mutant allele. (C) Predicted structure of the recombinant MK6a locus after Cre-mediated recombination. Cre-mediated recombination excises the neo-cassette from intron 1, thereby activating transcription of the mutant MK6a gene. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Strategy to generate inducible transgenic mice expressing the entire mutant HK6a gene. (A) Genomic locus of MK6a. (B) Targeting vector. MK6a exons and introns are replaced by corresponding mutant HK6a genomic sequence (blue). Asterisk represents the N171K mutation of HK6a. A neo-cassette is inserted in intron 1. Homologous recombination between the targeting vector and the MK6a genomic locus in embryonic stem (ES) cells is symbolized by X. (C) Predicted structure of the recombinant MK6a locus. The neo-cassette and its multiple polyA signals in intron 1 prevent gene transcription from the recombinant allele. (D) Predicted structure of the recombinant MK6a locus after Cre-mediated recombination. Cre-mediated recombination excises the neo-cassette from intron 1, thereby activating transcription of the mutant HK6a gene. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

10 Figure 9 Alternative strategy to generate inducible transgenic mice expressing the entire mutant HK6a gene. (A) Genomic locus of MK6a. (B) Targeting vector. MK6a exons 1–9 and introns in between are replaced by exon 1 containing N171K, the first intron and complementary DNA (cDNA) corresponding to HK6a exons 2–9 (blue). Asterisk represents the N171K mutation. A neo-cassette is inserted in intron 1. Homologous recombination between the targeting vector and the MK6a genomic locus in embryonic stem (ES) cells is symbolized by X. (C), Predicted structure of the recombinant MK6a locus. The neo-cassette and its multiple polyA signals in intron 1 prevents gene transcription from the recombinant allele. (D) Predicted structure of the recombinant MK6a locus after Cre-mediated recombination. Cre-mediated recombination excises the neo-cassette from intron 1, thereby activating transcription of the mutant HK6a gene. Journal of Investigative Dermatology Symposium Proceedings  , 37-46DOI: ( /j x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions