| First Author | Yu YE | Year | 1997 |
| Journal | Mouse Genome | Volume | 95 |
| Issue | 3 | Pages | 704-706 |
| Mgi Jnum | J:43273 | Mgi Id | MGI:1097453 |
| Citation | Yu YE, et al. (1997) Characterization of a transgene-induced mutant mouse line showing thymus atrophy and neuronal degeneration. Mouse Genome 95(3):704-706 |
| abstractText | Full text of Mouse Genome contribution: CHARACTERIZATION OF A TRANSGENE-INDUCED MUTANT MOUSE LINE SHOWING THYMUS ATROPHY AND NEURONAL DEGENERATION. Y. Eugene Yu1, George Stoica2, Asha S. Multani3, Wenguang Zhang1, Sen Pathak3,4, and Paul K.Y. Wong1. 1Science Park-Research Division, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957; Department of Veterinary Pathobiology, Texas A & M University, College Station, Texas 77843; 3Department of Cell Biology and 4Division of Laboratory Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030. The ts1 virus, a temperature-sensitive mutant of Moloney murine leukemia virus, can cause neuroimmunodegeneration in mice accompanied by neuronal loss, demyelination, and thymus atrophy (1). To study the consequence of expression of the ts1 virus envelope gene in activated T cells in vivo, we first constructed a ts1-env(H) transgene by linking the ts1 retroviral envelope gene to the 5'-flanking region of the human CGL-1/granzyme B gene (2) by multiple DNA-subcloning processes. We then generated 12 transgenic mouse lines by microinjecting the transgene construct into the pronuclei of mouse embryos (3). Of 108 offspring produced by sibling mating among transgenic mice from one of the transgenic lines, 22% exhibited a mutant phenotype: thymus atrophy and neuronal degeneration (Fig. 1 and Table 1). Those mutant mice were smaller than normal and could be recognized at about 10 days of age by their clasping of the hindlimbs when lifted by the tail. They also had an abnormal wobbly gait and shook when walking. The mice with the above phenotype inevitably died between 24 and 30 days of age. This transgenic mouse line was named TgN(Gbts1env)832Pkw (GB, granzyme B; ts1env, tsl-env) in accordance with the nomenclature rules for transgenic mice and will be abbreviated as TgN832Pkw. Quantitative Southern blot analysis of DNA isolated from transgenic mice demonstrated that hemizygous transgenic mice carry 7 to 8 copies of the transgene integrated in a head-to-tail orientation and that mice with the mutant phenotype have double the amount of transgene present in healthy hemizygous transgenic mice (data not shown). To conclusively establish the correlation between homozygosity for the transgene and the mutant phenotype, we constructed a genomic library using DNA isolated from presumably transgenic mice homozygous for the transgene and isolated genomic DNA sequences flanking the integrated transgene by using the transgene as a probe. Two overlapping clones containing both the transgene and mouse genomic DNA were obtained (Fig. 2A). A 1.8-kb EcoRI fragment of the genomic clones (Fig. 2A) was used as a probe in a Southern blot analysis to verify that the mice with mutant phenotype were homozygous for the transgene (Fig. 2B). Concordance between homozygosity for the 6.0-kb allele and the mutant phenotype has been confirmed in all 62 transgenic mice tested. No abnormalities were observed in any other transgenic lines carrying the ts1-env(H) construct except for mice from transgenic line EY47. In that line, a T(3;10) autosomal translocation was found to cosegregate with the transgene, and these genetic changes were then found to associate with the phenotype of impaired reproductive ability observed in EY47 mice (3). Fig. 1. (Legend). Thymus atrophy and mild to moderate motor neuronal degeneration observed in homozygous Tg832Pkw mice. (A) Thymus section from a nontransgenic littermate (X160); (B) thymus section from a homozygous TgN832Pkw transgenic mouse (X160); (C) section of the lumbar spinal cord from a homozygous TgN832Pkw transgenic mouse (X220). Table 1. Comparisons of body weight and ratio of thymus weight/body weight between homozygous TgN832Pkw transgenic mice and age-matched nontransgenic littermates or age-matched homozygous transgenic mice. Body weight (grams) +/- S.D.: Homozygous transgenic mice (n=9)**: 8.1 +/- 1.4; Hemizygous transgenic mice (n=15): 10.9 +/- 2.1(+); Nontransgenic littermates (n=11): 10.7 +/- 1.5(+). Ratio of thymu/body weight (x10-3) +/- S.D.: Homozygous transgenic mice (n=9)**: 3.1 +/- 1.3; Hemizygous transgenic mice (n=15): 7.5 +/- 1.5 (++); Nontransgenic littermates (n=11): 7.2 +/- 0.6 (++) * All mice used in the experiment were 19 days of age. ** No. of animals per group shown in parentheses. + Different from homozygous transgenic mice, p<0.005. ++ Different from homozygous transgenic mice, p<0.001. Fig. 2. (Legend). (A) Partial restriction-site map of genomic clones isolated from TgN832Pkw mouse genome containing both the transgene and mouse genomic DNAs. B, Bam HI site; R, EcoRI site. (B) Correlation between homozygosity of the transgene and the disease phenotype. A 1.8-kb EcoRI frgment isolated from TgN832Pkw genomic clones (see Fig. 2A) was used as a probe in a Southern analysis of Bam HI-digested genomic DNA isolated from a transgenic mouse with disease phenotype (lane 1), a transgenic mouse without disease phenotype (lane 2), and a nontransgenic mouse (lane 3). To map the chromosome location of the integrated transgene in TgN832Pkw mice, we employed the "interspecific mapping" approach. We began with Southern blot polymorphism screening using restriction enzyme-digested DNAs from C57BL/6J and Mus spretus. Using a 1.8-kb EcoRI fragment probe, one (8.0-kb) and two (5.1- and 2.9-kb) fraements were detected in Taq I-digested C57BL/6J and Mus spretus DNAs, respectively. Based on this result, we purchased a set of special Southern blotting filters from the Jackson Laboratory Backcross DNA Panel Mapping Resource. DNAs transferred to these filters were isolated from a panel of 94 backcross (C57BL/6JEI x SPRETUS/Ei)F1 x SPRETUS/Ei (over 2370 loci have been mapped in this BSS backcross panel [4]) and digested with Taq I prior to electrophoresis. The results of Southern hybridization of these membranes with the 1.8-kb EcoRI fragment probe showed that the 5.1- and 2.9-kb Mus spretus-specific fragments cosegregated. The segregation pattern of the 1.8-kb EcoRI fragment probe analyzed in the Jackson Laboratory Backcross DNA Panel Mapping Resource also showed 100% concordance to the microsatellite marker D19Mit11, which maps to chromosome 19 (Fig. 3). Rps4-rs6, Tdt, Nkx2.3, Pax2, and Tlx-1 are located 2.5 centimorgans or less from D19Mit11 which might be candidates for disruption by the transgene (5). However, the "knockout" mice with null mutation of the gene for Tdt or Pax2 did not exhibit the phenotype observed in homozygous TgN832Pkw mice (6, 7). Cytogenetic analysis of the karyotypes of the embryonic cells isolated from transgenic embryos revealed no visible abnormalities in any chromosomes (Fig. 4). Based on the mutant phenotype, we have named the transgene integration locus abnormal wobbly gait (awg). Several lines of immortalized cells have been established by infectinge primary transgenic embryonic fibroblasts with a defective retrovirus carrying the gene of the tsA58 T antigen (8). Fig. 3. (Legend). Mapping the transgene integration site in TgN832Pkw mice to chromosome 19 by interspecific backcross analysis. (A) The segregation patterns of 1.8-kb EcoRI fragment and linked genes in 94 backcross mice. Each column represents the chromosome identified in the backcross progeny that was inherited from (C57BL/6J x SPRETUS/Ei)Fl. The black and white boxes represent the presence of C57BL/6J and Mus spretus alleles, respectively. The number of animals inheriting each type of chromosome is given at the bottom. (B) A partial linkage map of chromosome 19 based on data in (A). Distances between loci in centimorgans are indicated at the right of the map. Fig. 4. (Legend). Giemsa-banded karyotype of a male TgN832Pkw transgenic embryonic fibroblast showing normal chromosome constitution. Chromosome analysis was conducted as described (12). Chromosome 19 pairs from two additional metaphases are arranged at bottom. To examine transgene expression, total cellular RNAs were isolated from cerebral cortex, brain stem, spinal cord, thymus, liver, spleen, testis, and ovary of mice from all 12 transgenic lines and subjected to ribonuclease protection assay (2). We could only detect the transgene mRNA in the thymus of TgN832Pkw mice (Fig. 5). Since the 5'-flanking region of the human CGL-1/granzyme B gene used in the transgene gene used in the transgene construct ts1-env(H) has been shown to target gene expression specifically to activated T cells isolated from the spleens of transgenic mice (2), the results shown in Fig. 5 suggest that the transgene flanking regions of the mouse chromosome in TgN832Pkw transgenic mice alters the tissue specificity of the transgene expression and cause the transgene to be exclusively expressed in the thymus (a similar observation was recently described in another line of transgenic mice and that transgene insertion was mapped to chromosome 17 [9]). Thus, the phenotype exhibited by homozygous TgN832Pkw transgenic mice is probably due to either one of two things: The first is expression of the transgene, i.e., the envelope gene of ts1 virus, from 14-16 copies of the transgene in homozygous transgenic mice (the phenomenon of transgene product causing the disease phenotype in homozygous but not hemizygous transgenic mice has been reported previously in transgenic mice harboring a mutant prion gene [10]). The second is the mutation generated by the presence of the transgene in the host genome, i.e., intragenic mutation or mutation that alters host gene expression through long-range effects on chromosome structure or by disrupting distal regulatory elements of a host gene (11). Fig. 5. (Legend). RNA analysis of transgene expression. In the assay, 10 ug of total RNA prepared from various organs was used. Lane 1, RNA from thymus of a ts1 virus-infected mouse; lane 2, RNA from thymus of a nontransgenic littermate; lanes 3-6, RNA from thymus, brain, spleen, and testes, respectively, of a hemizygous TgN832Pkw transgenic mouse. A 259-bp stretch of the probe (307-bp) was protected by ts1-env RNA. ACKNOWLEDGMENTS: P. Wong is the corresponding author. Transgenic mice were generated at our Science Park-Research Division transgenic mouse facility and the National Transgenic Development Facility at DNX, Inc., in Princeton, NJ. The authors thank Debra Hollowell, Wanda Fuller, and Yuhun Lu for technical assistance, Jo Peters for help with naming the transgene integration locus, and Jude Richard, in M.D. AndersonÕs Department of Scientific Publications for editorial assistance. This investigation was supported by Public Health Service Grants. 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