Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA
Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNAC. Huxley1,*, E. Passage2, A. Manson1, G. Putzu2,3, D. Figarella-Branger3, J. F. Pellissier3 and M. Fontés2,*
1Imperial College School of Medicine at St Mary's, London W2 1PG, UK, 2INSERM U406, Équipe No 3: Génome Humain et Développment, Faculté de Médecine de la Timone, BP 24, 13385 Marseille Cedex 5, France and 3Laboratoire de Pathologie Neuro-musculaire, Équipe DGRT No866, Faculté de Médecine de la Timone, 13351 Marseille Cedex 5, France
Received December 22, 1995;Revised and Accepted February 21, 1996
Construction of animal models of human inherited diseases is particularly important for testing gene therapy approaches. Towards this end, we constructed a mouse model for Charcot-Marie-Tooth disease type 1A by pronuclear injection of a YAC containing the human PMP22 gene. In one transgenic line, the YAC DNA is integrated in about eight copies and the PMP22 gene is strongly expressed to give a peripheral neuropathy closely resembling the human pathology. The disorder is dominant, causes progressive weakness of the hind legs, and there is severe demyelination in the peripheral nervous system including the presence of onion bulb formations. This approach will be valuable for pathologies produced by over-expression of a gene including trisomy and amplification in cancer. Such models will be particularly useful for testing gene therapy approaches if the transgene is human.
Charcot-Marie-Tooth disease (CMT) is the major form of hereditary peripheral neuropathy and causes significant neuromuscular impairment. The prevalence is about one affected individual in 2500, making it one of the most frequent inherited disorders. The first clinical signs of CMT usually appear during the second decade of life, and it is characterised by a progressive weakness of distal muscles, and hand and foot deformations. The disease is heterogeneous and is classically separated into a demyelinating form (CMT1) and an axonal form (CMT2). CMT1, the most frequent type, is characterised by a decrease in the nerve conduction velocity, due to the demyelination of the nerve fibers. The histology of the nerves is also characteristic and consists of a hypertrophic neuropathy with extensive demyelination of myelinated nerve fibers and `onion bulb' formations due to Schwann cell hyperplasia [for review of CMT, see Chance and Fischbeck, 1994 (1 )].
Subtype 1A, the most common form of the disease, is inherited as an autosomal dominant trait. This disease has been associated with the partial duplication of the sub-band 17p11.2 (2 ,3 ), spanning 1.5 Mb, and generally arising through unequal crossing over during male meiosis (3 -6 ). This led to the hypothesis that the disease phenotype is caused by over-expression of a gene due to gene dosage (2 ). Gene dosage, rather than disruption of a gene by the duplication, was confirmed by the finding of similar demyelinating neuropathies with low nerve conduction velocity in patients with other, cytologically visible, duplications of regions of 17p (7 -9 ).
It is now clear that the gene coding for the peripheral myelin protein-22 (PMP22) is responsible for the CMT1A phenotype. The human gene is duplicated in CMT1A patients (10 -13 ). In Trembler and Trembler-J mice mutations in the PMP22 gene are responsible for a phenotype resembling human CMT1A including hypomyelination of peripheral nerves, decrease in nerve conduction velocity, and muscle weakness (14 ,15 ). Also, Valentijn et al. have described a family with CMT1A where a point mutation in PMP22, identical to that found in the Trembler-J mouse, segregates with the disease and shows strong linkage to 17p11.2-p12 markers without the duplication (16 ). Finally, a case has been reported where a de novo point mutation has arisen in the PMP22 gene in a sporadic CMT1A case confirming the causative role of PMP22 in CMT1A. Gene dosage of PMP22 as the causative mechanism has been supported by the finding of increased levels of PMP22 mRNA in nerve biopsies from patients with CMT1A in relation to patients with other neuropathies (17 ,18 ) though the level of protein is reduced (19 ).
The CMT1A phenotype in humans most frequently results from a dosage effect caused by the duplication rather than point, or other mutations, of the PMP22 gene. In a number of different studies, the duplication has been found in 68% (6 ), 92% (20 ), 82% (21 ) and 91% (22 ) of families with CMT1. The duplication was also found in nine out of 10 de novo cases of CMT1 (23 ). Although the Trembler and Trembler-J mice are a model of CMT1A caused by point mutations, they are not a good model for CMT1A caused by over-expression of the PMP22 gene. A knock-out of the PMP22 gene has been reported recently but this again is not a model of the common form of CMT1A (24 ).
Thus, CMT1A is a unique case of an inherited partial trisomy, where the major (and maybe only) gene involved in the associated pathology, has been identified. Yeast artificial chromosomes are ideal for creating animal models for over-expression of genes because they contain very large regions of DNA which usually contain all the long range controlling elements which confer full levels of tissue specific expression (25 -27 ). This is in contrast to many transgenics made with minigene constructs where the transgene is often expressed at a very low level albeit in the correct tissues (28 ). Therefore, we decided to create a mouse model by introduction of a YAC which spans not only the PMP22 gene but a large proportion of the region duplicated in CMT1A patients.
We have used a 560 kb CEPH YAC (49G7, also called 49H7) which encompasses the proximal part of the CMT1A duplication (4 ,12 ,13 ,29 ,30 ) to make transgenic mice by pronuclear injection. The YAC carries the human PMP22 gene which spans about 40 kb (31 ) flanked by about 100 kb of upstream sequences and about 300 kb of downstream sequences (Fig. 1 a). The YAC DNA was gel purified and injected using modifications of previously published methods (32 ,25 ).
To check the integrity of the human transgene, we analysed first the human STS content of the mice. The six STSs shown in the YAC in Figure 1 a are spread throughout the YAC and were all found to be present in the mouse genome (data not shown) indicating that there is probably no big deletion in the integrated YAC. PFGE analysis of the PMP22 region was then carried out for two C22 mice (third and fourth generations). The observed ~100 kb SfiI fragment in the mice is of the same length as that of the control human DNA (Fig. 1 b), indicating that the YAC is largely intact and is the same as the human gene and is transmitted over generations without big rearrangements. This demonstrates that large exogenous fragments can be integrated and transmitted to descendants, without rearrangements, like the endogenous genome.
Using hybridization with a human PMP22 probe, we determined the number of copies integrated in the mouse genome. As a control, human DNA from an unaffected individual (two copies of PMP22), and a HNPP patient (one copy) were used. As can be seen in Fig. 1 c,d there are about eight copies of the YAC integrated in the mouse genome (the other strains have only integrated 1-2 copies, data not shown).
Fluorescence in situ hybridisation (FISH) was used to show that the YAC DNA is integrated at a single site which banding indicated was on either chromosome 8 or 12 (Fig. 3 , these two autosomes are very similar). The use of a chromosome 8 specific probe (a gift of C. Vourch) indicated that it is on chromosome 12. There are no known dominant mouse mutants with demyelinating neuropathies segregating with either chromosome 8 or 12 and it is rare for transgenes to have dominant effects due to the position of integration.
As the transgenic DNA is very large we expect to get high levels of tissue specific expression. PMP22 is known to be highly expressed in the Schwann cells of the peripheral nerves so the level of human mRNA was compared to the level of mouse mRNA in the sciatic nerve. Semi-quantitative RT-PCR using a pair of primers which amplify both the mouse and human cDNA was used to determine the relative level of expression of the human gene. The human PMP22 mRNA was found to be present in the sciatic nerves at about 1.7 times the level of the mouse mRNA (Fig. 4 a) indicating that each human gene (eight copies) is expressed at about 0.4 times the level of each mouse gene (two copies).
Histological examination is probably the best test to determine what pathology is causing the phenotype. Examination with light microscopy showed no changes in brain, cerebellum, brain stem, spinal cord, heart, lung, liver, and optic nerve. Muscle, however, showed the classical features of a denervation process and the sciatic nerve displayed widespread demyelination of medium to large axons without typical onion bulbs while small myelinated fibers appeared well preserved (Fig. 5 a). Some of the axons, either ensheathed by a thin myelin or demyelinated, were abnormally enlarged. There were no signs of acute axonal degeneration, no increase in endoneural connective tissue, and inflammatory cell infiltration was not present. Finally, the teasing technique was consistent with a primary demyelinating process.
Thus, we have constructed a mouse strain, suffering from a severe peripheral demyelinating neuropathy, which has most of the phenotypic traits in common with the clinical presentation of CMT1A. The phenotype appears several weeks after birth (about the same time as the appearance of the phenotype in Trembler mice), it is progressive, and preferentially affects the hindlegs. On histological examination, the only tissue to be affected is the peripheral nerves and here there is severe demyelination with occasional onion bulb formations. The high level of expression of the human PMP22 mRNA indicates that the phenotype is likely to be due to the expression of the extra copies of the human PMP22 gene, and that it is indeed perturbations in PMP22 expression that induce the demyelination.
The phenotype in the mice is more severe than CMT1A in that onset in humans is not until the 2nd decade of life and demyelination is not as severe in humans. In addition, there is occasional axonal hypertrophy in the mice, which is not the case in CMT1A. These differences could be due to the high level of expression of the PMP22 gene which is equivalent to an extra three copies of the gene rather than the one extra copy in most patients. A patient homozygous for the duplication has been reported (2 ) and is clinically more severe than normal with onset at less than 1 year, but the histology has not been described. The differences could also be due to it being a human gene which is being expressed rather than the mouse gene and this could be causing a dominant negative effect similar to point mutations rather than a dominant effect of over-expression found in most human patients.
More experiments will be needed to distinguish between these hypotheses. Analysis of the other mouse lines, which have integrated fewer copies (and their homozygous strains), will give information about the effect of varying amounts of human PMP22 expression. The introduction of a mouse YAC into transgenic mice, and comparison of the phenotype to that obtained with the human YAC will determine whether the human protein is acting differently to the mouse protein. The transgenic mice will also allow us to study the progression of the demyelination.
Using YAC DNA, we have created for the first time a mouse model of a human disease by over-expression of a human gene. The use of YAC DNA for making transgenic mice has the advantage that the gene of interest, in this case the PMP22 gene, is surrounded by a large amount of surrounding DNA. Just eight copies of the transgene was enough to give 1.7 times as much expression as the two mouse genes and the tissue specificity of expression followed that of the mouse gene. Irrespective of the exact mechanism of pathological action of the human PMP22 protein, these mice will be a valuable tool to test therapeutic approaches which are aimed at reducing the level of expression of the human gene.
Such transgenic mice with high levels of expression of human genes, either normal or modified, from YAC DNA should have many applications in studying gene expression and making mouse models in the context of gene therapy. It is also noteworthy that as the transgene is human it will be possible to use the same tools in human somatic cell gene therapy as in the mouse model.
YAC DNA was isolated from preparative pulsed field gels using a modification of a previously described method (32 ). After treatment with agarase the DNA solution was concentrated about 2 fold with a Millipore Ultrafree-MC 30,000 NMWL Filter Unit (Millipore Cat # UFC3 TTK 00) and then dialysed for several hours on a Millipore filter (cat no. VMW 02500, type VM, pore size 0.05 [mu]m) against microinjection buffer (10 mM Tris pH 7.4, 0.2 mM EDTA, 100 mM NaCl). Transgenic mice were generated by standard techniques of pronuclear injection (36 ) using C57BL/6J * CBA/Ca F1 mice as donors. Subsequent crosses were to the same F1 mice.
For the FISH analysis, Alu PCR products from the YAC 49G7 were synthesised and labelled as described elsewhere (37 ). A quantity of 100 ng of labelled probe was preannealed to 20 fold excess of Cot 1 DNA for 20 min at 37oC and then precipitated in ethanol and centrifuged. The pellet was resuspended in 10 [mu]l and hybridized to denatured chromosome spreads at 37oC overnight in 50% formamide. The probe sequences were detected with avidin-FITC (Sigma). Slides were examined on a Zeiss Axiophot microscope equipped with a 3 CCD camera.
The YAC 49G7, also called 49H7, from the CEPH library (38 ) has been previously described (4 ,12 ,13 ,29 ,30 ). Mouse DNA was extracted from cultured fibroblasts using standard procedures both as low molecular weight DNA for analysis of copy number, and as high molecular weight DNA in agarose blocks. Blocks were digested with 20 U of SfiI overnight. Pulse field gel electrophoresis was performed on a BioRad CHEF DRII with a pulse time of 10 s to 45 s over 33 h at 160 V. The gels were blotted on PALL membranes in 10* SSC overnight. The probe was labelled using random priming and stringent washes were performed at 0.1* SSC at 65oC. The signal was imaged and quantitated with a Phosphorimager. The exon 2 probe does cross hybridise to the mouse gene but the sizes of the HindIII and SfiI fragments are different in mouse than human DNA.
Up to 200 mg of tissue was snap frozen in liquid nitrogen for storage at -80oC for up to 3 weeks. PolyA+ RNA was extracted using the Invitrogen Micro-Fast Track kit according to the manufacturer's protocol. A quantity of 10 to 150 ng of polyA+ RNA were used for cDNA synthesis using the Invitrogen cDNA cycle kit.
For comparison of the levels of human and mouse mRNA, cDNA was amplified using two primers; 5' GTC TCC ACC/G ATC GTC AGC CAA TG 3' (starts at position 249 of accession D11428) and 5' CTC ATC ACG CAC AGA CCA GCA AG 3' (starts position 523 of accession D11428) which are homologous to both the mouse and the human cDNAs. The PCR product crosses an intron and does not amplify from genomic DNA. The 275 bp human product cuts with TaqI to give two fragments of 154 and 121 bp and the mouse product cuts with AluI to give two fragments of 198 and 77 bp. Amplification was for 30 cycles and the system was found to give the same ratio of mouse to human with 24 to 32 cycles of PCR. 33P was incorporated during the PCR and the 2% agarose gel was dried and quantitated using a Phosphorimager (Molecular Dynamics, `Imagequant' programme).
For section b, three primers were used in the PCR reaction; 5' CTC TTG TTG GGG ATC CTG 3' (mouse specific, starts at position 196 of sequence accession number M32240), 5' CTT CCT CAG GAA ATG TCC 3' (human specific, starts position 322 of sequence accession number D11428) and 5' CAG ACC AGC AAG A/GAT TTG 3' (both, starts at position 512 of sequence accession number D11428). The PCR product crosses an intron and there is no product from genomic DNA. A PCR assay for mouse Hprt cDNA was used to confirm the presence of good quality cDNA in each sample. This system is not quantitative but the intensities of the bands does reflect the amount of specific mRNA present.
Samples of brain, cerebellum, brainstem, spinal cord, heart, lung, liver, hind leg muscle, and optic and sciatic nerves from transgenic mice were processed for light and electron microscopy.
For light microscopy, samples of brain, cerebellum, and brainstem were fixed in 10% formol, paraffin embedded and stained with hematoxylin-eosin (HE), Loyer method and luxol-periodic acid-Schiff-fast blue. Samples of spinal cord, heart, lung, and liver were fixed in 4% paraformaldehyde, then in 8% paraformaldehyde and stained with HE. Muscle specimens were flash-frozen in isopentane pre-cooled in liquid nitrogen and processed for histology and histoenzymology. Standard staining techniques were used as previously reported (39 ). Optic and sciatic nerves were fixed in 2.5% glutaraldehyde and embedded in Araldite. Semi-thin sections were stained with hematoxylin-phloxin-saffron (HPS) and paraphenylene diamine (PPD). In addition, a sample of sciatic nerve stained with osmium tetroxide was placed in 66% glycerine for 48 h, then transferred in 100% glycerine for teasing technique.
For electron microscopy, sciatic nerve was fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium and embedded in Araldite. Ultra-thin sections were double-stained with uranyl and lead citrate.
We thank Dr Feuerstein for his help in mouse dissection, Gail Baker for caring for the mice, and Bob Williamson, and Phil Avner for discussions. This work has been supported by AFM (Association Française contre les myopathies).
1 Chance, P.F. and Fischbeck, K.H. (1994) Molecular genetics of Charcot-Marie-Tooth disease and related neuropathies. Hum. Mol. Genet., 3, 1503-1507.MEDLINE Abstract
2 Lupski, J.R., Montes de Oca-Luna, R., Slaugenhaupt, S., Pentao, L., Guzzetta, V., Trask, B.J., Saucedo-Cardenas, O., Barker, D.F., Killian, J.M., Garcia, C.A., Chakravarti, A. and Patel, P.I. (1991) DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell, 66, 219-232.MEDLINE Abstract
3 Raeymaekers, P., Timmerman, V., Nelis, E., De Jonghe, P., Hoogendijk, J.E., Baas, F., Barker, D.F., Martin, J.-J., De Visser, M., Bolhuis, P.A. and Van Broeckhoven, C. (1991) Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT 1a). The HMSN Collaborative Research Group. Neuromuscul. Disord., 1, 93-97.MEDLINE Abstract
4 Pentao, L., Wise, C.A., Chinault, A.C., Patel, P.I. and Lupski, J.R. (1992) Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit. Nature Genet., 2, 292-300.MEDLINE Abstract
5 Palau, F., Löfgren, A., De Jonghe, P., Bort, S., Nelis, E., Sevilla, T., Martin, J.-J., Vilchez, J., Prieto, F. and Van Broeckhoven, C. (1993) Origin of the de novo duplication in Charcot-Marie-Tooth disease type 1A: unequal nonsister chromatid exchange during spermatogenesis. Hum. Mol. Genet., 2, 2031-2035.MEDLINE Abstract
6 Wise, C.A., Garcia, C.A., Davis, S.N., Heju, Z., Pentao, L., Patel, P.I. and Lupski, J.R. (1993) Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMT1A duplication. Am. J. Hum. Genet., 53, 853-863.MEDLINE Abstract
7 Chance, P.F., Bird, T.D., Matsunami, N., Lensch, M.W., Brothman, A.R. and Feldman, G.M. (1992) Trisomy 17p associated with Charcot-Marie-Tooth neuropathy type 1A phenotype: Evidence for gene dosage as a mechanism in CMT1A. Neurology, 42, 2295-2299.MEDLINE Abstract
8 Lupski, J.R., Wise, C.A., Kuwano, A., Pentao, L., Parke, J.T., Glaze, D.G., Ledbetter, D.H., Greenberg, F. and Patel, P.I. (1992) Gene dosage is a mechanism for Charcot-Marie-Tooth disease type 1A. Nature Genet., 1, 29-33.MEDLINE Abstract
9 Roa, B.B., Garcia, C.A., Wise, C.A., Anderson, K., Greenberg, F., Patel, P.I. and Lupski, J.R. (1993) In C.G. Epstein (ed.), Phenotypic mapping of Down Syndrome and other aneuploid conditions. Wiley-Liss, Inc., New York, Vol.384, pp. 187-205.
10 Matsunami, N., Smith, B., Ballard, L., Lensch, M.W., Robertson, M., Albertsen, H., Hanemann, C.O., Müller, H.W., Bird, T.D., White, R. and Chance, P.F. (1992) Peripheral myelin protein-22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot-Marie-Tooth 1A. Nature Genet., 1, 176-179.MEDLINE Abstract
11 Patel, P.I., Roa, B.B., Welcher, A.A., Schoener-Scott, R., Trask, B.J., Pentao, L., Snipes, G.J., Garcia, C.A., Francke, U., Shooter, E.M., Lupski, J.R. and Suter, U. (1992) The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A. Nature Genet., 1, 159-165.MEDLINE Abstract
12 Timmerman, V., Nelis, E., Van Hul, W., Nieuwenhuijsen, B.W., Chen, K.L., Wang, S., Othman, K.B., Cullen, B., Leach, R.J., Hanemann, C.O., De Johghe, P., Raeymaekers, P., van Ommen, G.-J.B., Martin, J.-J., Müller, H.W., vance, J.M., Fischbeck, K.H. and Van Broeckhoven, C. (1992) The peripheral myelin protein gene PMP-22 is contained within the Charcot-Marie-Tooth disease type 1A duplication. Nature Genet., 1, 171-175.MEDLINE Abstract
13 Valentijn, L.J., Bolhuis, P.A., Zorn, I., Hoogendijk, J.E., van den Bosch, N., Hensels, G.W., Stanton Jr., V.P., Housman, D.E., Fischbeck, K.H., Ross, D.A., Nicholson, G.A., Meershoek, E.J., Dauwerse, H.G., van Ommen, G.-J.B. and Baas, F. (1992) The peripheral myelin gene PMP-22/GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A. Nature Genet., 1, 166-170.MEDLINE Abstract
14 Suter, U., Moskow, J.J., Welcher, A.A., Snipes, G.J., Kosaras, B., Sidman, R.L., Buchberg, A.M. and Shooter, E.M. (1992) A leucine-to-proline mutation in the putative first transmembrane domain of the 22-kDa peripheral myelin protein in the trembler-J mouse. Proc. Natl Acad. Sci. USA, 89, 4382-4386.MEDLINE Abstract
15 Suter, U., Welcher, A.A., Ozcelik, T., Snipes, G.J., Kosaras, B., Francke, U., Billings-Gagliardi, S., Sidman, R.L. and Shooter, E.M. (1992) Trembler mouse carries a point mutation in a myelin gene. Nature, 356, 241-244.MEDLINE Abstract
16 Valentijn, L.J., Baas, F., Wolterman, R.A., Hoogendijk, J.E., van den Bosch, N.H.A., Zorn, I., Gabreëls-Festen, A.A.W.M., de Visser, M. and Bolhuis, P.A. (1992) Identical point mutations of PMP-22 in trembler-J mouse and Charcot-Marie-Tooth disease type 1A. Nature Genet., 2, 288-290.MEDLINE Abstract
17 Kamholz, J., Shy, M. and Scherer, S. (1994) Elevated expression of messenger RNA for peripheral myelin protein 22 in biopsied peripheral nerves of patients with Charcot-Marie-Tooth disease type 1A. Ann. Neurol., 36, 451-452.MEDLINE Abstract
18 Yoshikawa, H., Nishimura, T., Nakatsuji, Y., Fujimura, H., Himoro, M., Hayasaka, K., Sakoda, S. and Yanagihara, T. (1994) Elevated expression of messenger RNA for peripheral myelin protein 22 in biopsied peripheral nerves of patients with Charcot-Marie-Tooth disease type 1A. Ann. Neurol., 35, 445-450.MEDLINE Abstract
19 Hanemann, C.O., Stoll, G., D'Urso-D., Fricke-W., Martin, J.J., Van Broeckhoven, C., Mancardi, G.L., Bartke, I. and Muller, H.W. (1994) Peripheral myelin protein-22 expression in Charcot-Marie-Tooth disease type 1a sural nerve biopsies. J. Neurosci. Res., 37, 654-659.MEDLINE Abstract
20 Ionasescu, V.V., Ionasescu, R. and Searby, C. (1993) Screening of dominantly inherited Charcot-Marie-Tooth neuropathies. Muscle Nerve, 16, 1232-1238.MEDLINE Abstract
21 Mostacciuolo, M.L., Schiavon, F., Angelini, C., Miccoli, B., Piccolo, F. and Danieli, G.A. (1995) Frequency of duplication at 17p11.2 in families of northeast Italy with Charcot-Marie-Tooth disease type 1. Neuroepidemiology, 14, 49-53.MEDLINE Abstract
22 Ionasescu, V.V. (1995) Charcot-Marie-Tooth neuropathies: from clinical description to molecular genetics. Muscle Nerve, 18, 267-275.MEDLINE Abstract
23 Hoogendijk, J.E., Hensels, G.W., Gabreëls-Festen, A.A.W.M., Gabreëls, F.J.M., Janssen, E.A.M., de Jonghe, P., Martin, J.-J., van Boeckhoven, C., Valentijn, L.J., Baas, F., de Visser, M. and Bolhuis, P.A. (1992) De-novo mutation in hereditary motor and sensory neuropathy type 1. Lancet, 339, 1081-1082.MEDLINE Abstract
24 Adlkofer, K., Martini, R., Aguzzi, A., Zielasek, J., Toyka, K.V. and Suter, U. (1995) Hypermyelination and demyelinating peripheral neuropathy in Pmp22-deficient mice. Nature Genet., 11, 274-280.MEDLINE Abstract
25 Peterson, K.R., Clegg, C.H., Huxley, C., Josephson, B.M., Haugen, H.S., Furukawa, T. and Stamatoyannopoulos, G. (1993) Transgenic mice containing a 248 kb yeast artificial chromosome carrying the human [beta]-globin locus display proper developmental control of human globin genes. Proc. Natl Acad. Sci. USA, 90, 7593-7597.MEDLINE Abstract
26 Schedl, A., Montoliu, L., Kelsey, G. and Schutz, G. (1993) A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature, 362, 258-261.MEDLINE Abstract
27 Huxley, C. (1994) In J.K. Setlow (ed.), Genetic Engineering. Plenum Press, New York, Vol.16, pp. 65-91.
28 Jaenisch, R. (1988) Transgenic animals. Science, 240, 1468-1474.MEDLINE Abstract
29 Nieuwenhuijsen, B.W., Chen, K.L., Chinault, A.C., Wang, S., Valmiki, V.H., Meershoek, E.J., van Ommen, G.J.V. and Fischbeck, K.H. (1992) A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region. Hum. Mol. Genet., 1, 605-612.MEDLINE Abstract
30 Chevillard, C., Le Paslier, D., Passage, E., Ougen, P., Billault, A., Boyer, S., Mazan, S., Bachellerie, J.P., Vignal, A., Cohen, D. and Fontes, M. (1993) Relationship between Charcot-Marie-Tooth 1A and Smith-Magenis regions. snU3 may be a candidate gene for the Smith-Magenis syndrome. Hum. Mol. Genet., 2, 1235-1243.MEDLINE Abstract
31 Suter, U., Snipes, G.J., Schoener-Scott, R., Welcher, A.A., Pareek, S., Lupski, J.R., Murphy, R.A., Shooter, E.M. and Patel, P.I. (1994) Regulation of tissue-specific expression of alternative peripheral myelin Protein-22 (PMP22) gene transcripts by two promoters. J. Biol. Chem., 269, 25795-25808.MEDLINE Abstract
32 Gnirke, A., Huxley, C., Peterson, K. and Olson, M.V. (1993) Microinjection of intact 200- to 500 kb fragments of YAC DNA into mammalian cells. Genomics, 15, 659-667.MEDLINE Abstract
33 Rudnik-Schöneborn, S., Röhrig, D., Nicholson, G. and Zerres, K. (1993) Pregnancy and delivery in Charcot-Marie-Tooth disease type 1. Neurology, 43, 2011-2016.MEDLINE Abstract
34 Spreyer, P., Kuhn, G., Hanemann, C.O., Gillen, C., Schaal, H., Kuhn, R., Lemke, G. and Müller, H.W. (1991) Axon-regulated expression of a Schwann cell transcript that is homologous to a `growth arrest-specific' gene. EMBO J., 10, 3661-3668.MEDLINE Abstract
35 Welcher, A.A., Suter, U., De Leon, M., Snipes, G.J. and Shooter, E.M. (1991) A myelin protein is encoded by the homologue of a growth arrest-specific gene. Proc. Natl Acad. Sci. USA, 88, 7195-7199.MEDLINE Abstract
36 Hogan, B., Costantini, F. and Lacy, E. (1986) Manipulating the mouse embryo. Cold Spring Harbor.
37 Lengauer, C., Green, E.D. and Cremer, T. (1992) Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification. Genomics, 13, 826-828.MEDLINE Abstract
38 Albertsen, H.M., Abderrahim, H., Cann, H.M., Dausset, J., Le Paslier, D. and Cohen, D. (1990) Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc. Natl Acad. Sci. USA, 87, 4256-4260.MEDLINE Abstract
39 Dubowitz, V. and Brooke, M.H. (1973) In W.B. Saunders (ed.), Muscle Biopsy. A Modern Approach. Philadelphia, P.A., pp.
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