摘要
DFNA64是本课题组在一个常染色体显性遗传非综合征型耳聋家系中定位的新的耳聋基因,通过定位区域已知基因直接测序找到与耳聋表型共分离的DIABLO基因c.377C>T杂合突变。DIABLO是线粒体内膜上非常重要的细胞凋亡促进蛋白,本课题组研究提示p.S126L突变体对于外界刺激更加敏感,可引发线粒体膜电位慢性损伤。进一步的研究需要在动物模型中复制人类的临床表现,进而验证和分析突变型DIABLO对内耳毛细胞的损伤作用,从而揭示致病基因突变导致耳聋的机理、机制,为预防和治疗的可能性打下基础。
一.DFNA64转基因小鼠模型的建立与鉴定
本研究利用哺乳动物高效表达载体pcDNA3.1 (+)构建包含CMV启动子、人工构建内含子、目的基因cDNA编码序列、多聚腺苷酸信号等元件的转基因载体,通过原核DNA显微注射转基因技术将构建的外源转基因载体DNA片段注射到CDl远交系小鼠合子的前核中,分别建立并得到了整合有突变型和野生型Diablo基因序列的DFNA64转基因小鼠模型；利用PCR鉴定小鼠的基因型；原代转基因阳性小鼠自交建立多个转基因品系及纯合子转基因小鼠；real-time PCR检测Diablo基因在神经系统组织中的表达量,证实Diablo基因在DFNA64转基因小鼠体内的过量表达。
二.DFNA64基因敲入小鼠模型的建立
本研究利用定点同源重组原理和基于ES细胞的基因打靶技术建立DFNA64基因敲入小鼠模型,在129品系ES细胞基因组中置入与人类DFNA64突变同源的p.S124L突变,获得嵌合体小鼠。载体的设计采用一长一短两段目的基因的同源序列,同源序列之间插入突变序列和loxP序列锚定的正选择筛选的neo基因,将线性化的载体DNA电转至ES细胞,通过定点同源重组整合到ES细胞基因组序列,利用抗生素G418和长片段PCR联合筛选,剔除未整合和随机插入载体序列的ES细胞,筛选到阳性的ES细胞经显微注射进入宿主囊胚,体外培养后移植到同步化假孕小鼠,发育为嵌合体小鼠。DFNA64小鼠模型的建立,为DIABLO基因功能研究、致聋机制、发病前预防、药物筛选等后续研究工作提供平台。
A new DFNA locus named DFNA64, was mapped in an autosomal dominant non-syndromic hearing loss family. By the means of direct sequencing of candidate genes in mapped region, heterozygous missense mutation c.377C>T was identified in DIABLO gene, which was segregated with the disease phenotype. DIABLO plays a key role in the promotion of apoptosis by binding the inhibitor of apoptosis protein. The previous investigations suggest mutant DIABLO while retaining the pro-apoptotic function, triggers significant degradation of both wildtype and mutant Smac/Diablo, rendering host mitochondria susceptible for calcium-induced membrane potential loss.. Mutant mouse models are necessary to understand the mechanism of inner ear hair cells damage by mutant DIABLO.
1. Creation and identification of DFNA64 transgenic mouse model
In this study, we obtained two lines of transgenic mouse models expressing wild and mutant diablo respectively by microinjection of linearized plasmid construct into fertilized eggs of CD1 strain mice. The construct is derived from pcDNA3.1(+), which is designed for high-level stable and transient expression in mammalian hosts. It contains CMV promotor, mutant or wild cDNA fragment of diablo, artifical intron and bGH-polyA signal. After identified the genotype of founders, generate transgenic mouse by cross the founders. Real-time PCR identified the overexpression of both wild and mutant diablo in nervous system.
2.Creation of DFNA64 knock-in mouse model
We also established knock-in mouse model by gene targeting. Targeting ES cells from 129 strain mice by electro-transformation, with construct consisting of two homologous sequences with floxed neo castle and mutation inserted. The targeted ES cells were screened out by antibiotic G418 and PCR reaction, then microinjected into embryos. We obtained five chimera mice. DFNA64 mouse models play significant roles in the study into gene function of DIABLO, mechanism of hearing loss, prevention before onset, therapeutic approaches.
引文
1. Lin T. P. Microinjection of mouse eggs. Science.1996,151:333-337
2. Gordon J. W., Scangos G. A., Plotkin D. J., et al. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc.Natl.Acad.Sci.1980,77:7380-7384
3. Gordon J.W., Ruddle F.H. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science. 1981,214:1244-1246
4. Brinster R. L., Chen H. Y., Trumbauer M. E., et al. Factors affecting the efficiency of introducing foreign DNA into mice by microinjected into mouse eggs. Proc. Natl. Acad. Sci.1985, 82:4438-4442
5乔德瑞,朱芷葳.基因打靶技术及其前景应用.现代农业科技.2009,20
6 Doetschman T., et al. Nature.1987,330:57657-57658.
7. Brinster R. L., Chen H. Y., Trumbauer M. E., et al. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell.1981,27:223-231
8. Wilkie T.M., Brinster R.L., Palmiter R. D. Germline and somatic mosaicism in transgenic mice. Dev. Biol.1986,118:9-18
9. Sandgren E. P., Palmiter R. D., Heckel J. L., et al. Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell.1991,66:245-256
10. Grosveld F., van Assendelft R., Greaves D.R., et al. Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell.1987,51:975-985
11.杨晓,黄培堂等.基因打靶技术.北京：科学出版社,2002
12. Sternberg N., Hamilton D. Bacteriophage P1 site-specific recombination. Ⅰ. Recombination between loxP sites. J Mol Biol. 1981,150:467-486
13. Simpson E. M., Linder C. C., Sargent E. E., et al. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat Genet.1997,16:19-27
14. Lemckert F. A., Sedgwick J. D., Korner H. Gene targeting in C57BL/6 ES cells successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro. Nucleic Acids Res.1997,25:917-918
15. Brook F. A., Gardner R. L. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci.1997, 94:5709-5712.
16. Erway L.C., Zheng Q. Y., Johnson K. R. Inbred strains of mice for genetics of hearing in mammals:searching for genes for hearing loss, in:J. F. Willott (Ed.), Handbook of Mouse Auditory Research:from Behavior to Molecular Biology, CRC Press,2001
17. Willott J. F., Tanner L.,O'Steen J., et al. Acoustic startle and prepulse inhibition in 40 inbred strains of mice. Behav. Neurosci.2003,117:716-727.
18. Joyner A. L. Gene Targeting:A Practical Approach, Oxford Univ. Press, Oxford, NY,2000.
19. Gao J. G., Wu X. D., Zuo J. Targrting hearing genes in mice. Molecular Brain Research.2004,132:192-207
20.Nagy A., Gertsenstein M., Vintersten K., et al. Manipulating the mouse embryo:A laboratory manual. Cold Spring Harbor Laboratory Press 2003.
21. Pinkert C. A. Transgenic animal technology:A laboratory Handbook. Elsevier Inc. Press 2003.
22. Friedman L. M., Dror A. A., Avraham K. B. Mouse models to study inner ear development and hereditary hearing loss. Int J Dev Biol.2007,51:609-631
22. Brinster R. L., Allen J. M., Behringer R. R., et al. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci.1988,85:836-840
23. Gu H., Zou Y. R., Rajewsky K. Independt control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell.1993,73:1155
24. Mansour A. Y., Lou M. A., Ahmed A., et al. The antireflux feeding gastrostomy:an improved method. J Natl Med Assoc.1988, 80:1006-1008
1. http://www.jax.org
2. Gao J. G., Wu X. D., Zuo J. Targrting hearing genes in mice. Molecular Brain Research.2004,132:192-207
3. Anagnostopoulos A. V. A compendium of mouse knockouts with inner ear defects. Trends Genet.2002,18:499
4. Hubbard T., Barker D., Birney E., et al. The Ensembl genome database project. Nucleic Acids Res.2002,30:38-41.
5. Zuo J. Transgenic and gene targeting studies of hair cell function in mouse inner ear. J Neurobiol.2002,53:286-305.
6. Friedman L. M., Dror A. A., Avraham K. B. Mouse models to study inner ear development and hereditary hearing loss. Int J Dev Biol.2007,51:609-631
7. Gabriel H. D., Jung D., Butzler C., et al. Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice. J Cell Biol.1998,140:1453-1461.
8. Gu H., Marth J. D., Orban P. C., et al. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science.1994,265:103
9. Nagy A. Cre recombinase:the universal reagent for genome tailoring. Genesis.2000,26:99
10. Lakso M., Sauer B., Mosinger B., et al. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci.1992,89:6232
11.Cohen-Salmon M., Ott T., Michel V., et al Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr Biol.2002, 12:1106-1111
12. Pirvola U., Ylikoski J., Trokovic R., et al FGFR1 is required for the development of the auditory sensory epithelium. Neuron. 2002,35:671-680
13. Matsuoka T., Ahlberg P. E., Kessaris N., et al. Neural Crest Origins of the Neck and Shoulder. Nature.2005,436:347-355
14. Lecureuil C., Fontaine I., Crepieux P., et al. Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice. Genesis.2002,33:114-118
15. Rickheit et al. Endocochlear potential depends on Cl- channels: mechanism underlying deafness in Bartter syndrome IV. Embo J. 2008,27:2907
16. Barrionuevo F., Naumann A., Bagheri-Fam S., et al. Sox9 is required for invagination of the otic placode in mice. Dev Biol. 2008,317:213-224.
17. Schultz et al. Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39. Am J Hum Genet.2009,85:25
18. Perrin et al. β-actin and γ-actin are each dispensable for auditory hair cell development but required for Stereocilia maintenance. PLoS Genetics.2010,6(10)
19. Chen and Nathans Estrogen-related receptor beta/NR3B2 controls epithelial cell fate and endolymph production by the stria vascularis. Dev Cell.2007,13:325.
20. Haugas et al. Gata2 is required for the development of inner ear semicircular ducts and the surrounding perilymphatic space. Dev Dyn.2010,239:2452
21. Jones et al. Ciliary proteins link basal body polarization to planar cell polarity regulation. Nat Genet.2008,40:69
22. Lobe C. G., Koop K. E., Kreppner W., et al. Z/AP, a double reporter for cre-mediated recombination. Dev Biol. 1999,208:281-292.
23. Novak A., Guo C., Yang W., et al. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis.2000,28:147-155
24. Shi Y. Mammalian RNAi for the masses. Trends Genet.2003,19:9-12
25. Tang L. S., Montemayor C., Pereira F. A. Sensorineural hearing loss:potential therapies and gene targets for drug development. IUBMB Life.2006,58:525-530.
26. Robertson N. G., Khetarpal U., Gutoerrez-Espeleta G. A., et al. Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening. Geniemics.1994,23:42-50
27. Klockars T., Perheentupa T., Dahl H. H. In silico analyses of mouse inner-ear transcripts. J Assoc Res Otolaryngol.2003, 4:24-40
28. Chen Z. Y., Corey D.P. Understanding inner ear development with gene expression profiling. J Neurobiol.2002,53:276-285
29. Zheng J., Shen W., He D. Z., et al. Prestin is the motor protein of cochlear outer hair cells. Nature.2000,405:149-155
30. Crozet F., el Amraoui A., Blanchard S., et al. Petit, Cloning of the genes encoding two murine and human cochlear unconventional type Ⅰ myosins. Genomics.1997,40:332-341
31.Harter C., Ripoll C., Lenoir M., et al. Expression pattern of mammalian cochlea outer hair cell (OHC) mRNA:screening of a rat OHC cDNA library. DNA Cell Biol.1999,18:1-10
32. Dulon D., Luo L., Zhang C., et al. Expression of small-conductance calcium-activated potassium channels (SK) in outer hair cells of the rat cochlea. Eur J Neurosci.1998,10:907-915
33. Ahn K., Crenshaw Ⅲ E. Generation of conditional mutants in the mouse neural tube and inner ear using gene regulatory elements from the Brn4/Pou3f4 gene.2001 ARO Abstract,2001
34. Ahn K., Samadi D., Saunders J., et al. Genetic and functional analyses of stapes malformations in the brn4/pou3f4 mouse mutant.2004 ARO Abstract,2004
35. Sage C., Huang M., Karimi K., et al. Identification of a key regulator, the retinoblastoma gene(Rb), in hair cell development.2004 ARO Abstract,2004
36. Hebert J.M., McConnell S.K. Targeting of cre to the Foxgl (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures. Dev Biol.2000,222:296-306
37. Ohyama T., Groves A.K. Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome. Genesis. 2004,38:195-199.
38. Bouchard M., Souabni A., Busslinger M. Tissue-specific expression of ere recombinase from the Pax8 locus. Genesis. 2004,38:105-109
39. Tian Y., Li M., Fritzsch B., et al. Creation of a transgenic mouse for hair-cell gene targeting by using a modified bacterial artificial chromosome containing Prestin. Dev Dyn. 2004,231:199-203
40. Li M., Tian Y., Fritzsch B., et al. An inner hair cell-Cre expressing transgenic mouse. Genesis.2004,39:173-177
41.Matei V., Pauley S., Kaing S., et al. Smaller inner ear sensory epithelia in Neurog 1 null mice are related to earlier hair cell cycle exit. Dev Dyn.2005,234:633-650
42. Coghill E. L., Hugill A., Parkinson N. et al. A gene-driven approach to the identification of ENU mutation in the mouse. Nat Genet.2002,30:255-256
43. Kudo T., Kure S., Ikeda K., et al. Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness. Human Molecular Genetics. 2003,12:995-1004
44. Inoshita A., Lizuka T., Okamura H.O., et al. Postnatal development of the organ of Corti in dominant-negative Gjb2 transgenic mice. Neuroscience.2008,156:1039-1047
45. Estevez R., Boettger T., Stein V., et al. Barttin is a Cl--channel β-subunit crucial for renal Cl--reabsorption and inner ear K+-secretion. Nature.2001,414:558-561