A single point mutation in a novel immune-associated nucleotide gene 5 (Ian5) coincides with severe T cell lymphopenia in BB rats. We used a transgenic rescue approach in lymphopenic BB-derived congenic F344.lyp/lyp rats to determine whether this mutation is responsible for lymphopenia and to establish the functional importance of this novel gene. A 150-kb P1 artificial chromosome (PAC) transgene harboring a wild-type allele of the rat Ian5 gene restored Ian5 transcript and protein levels, completely rescuing the T cell lymphopenia in the F344.lyp/lyp rats. This successful complementation provides direct functional evidence that the Ian5 gene product is essential for maintaining normal T cell levels. It also demonstrates that transgenic rescue in the rat is a practical and definitive method for revealing the function of a novel gene.
the biobreeding (BB) strain of rats develops spontaneous type 1 diabetes, which is closely linked to T cell lymphopenia. Diabetes pathology in this strain closely resembles human diabetes (6). Therefore, susceptibility genes for diabetes found in this strain can be considered as potential candidate genes in humans. In this strain, we linked the peripheral T cell lymphopenia to a 100-kb locus on chromosome 4 (10, 13, 14). To study the effects of the Lyp gene in the absence of diabetes, we generated congenic F344.lyp/lyp rats, in which the T cell lymphopenia locus from an inbred diabetes-prone BB strain was introgressed onto the genome of diabetes-resistant F344 rats (17). This approach led to identification of the lymphopenia locus and subsequent cloning of a novel immune-associated nucleotide gene 5 (Ian5) (9, 13). We have established that lymphopenic rats lack one C nucleotide in exon 3 of the Ian5 gene. This frame-shift mutation in the presumed open reading frame results in a significantly truncated protein product. Subsequently, it has been reported that the Ian5 gene has sequence similarity to a novel and largely uncharacterized protein family distinguished by a well-conserved GTP-binding motif and is involved in the function of the immune system (2, 4). The sequence of this novel gene is well preserved in plants, mice, rats, and humans, indicating involvement in fundamental defense mechanisms (20–22).
The aim of the present study was to use a transgenic approach to directly verify that the T cell lymphopenia in the F344.lyp/lyp rats would be corrected by restoring a functional copy of the Ian5 gene. This would indicate that the lymphopenia is caused by the frame-shift mutation of the Ian5 gene and would establish the functional importance of this novel gene. To accomplish this, pronuclei of fertilized eggs from a T cell lymphopenic F344.lyp/lyp congenic strain were microinjected with a ∼150-kb genomic clone containing the wild-type allele of the Ian5 gene from a Brown Norway (BN) rat. The resulting transgenic offspring were examined for expression of Ian5 and for symptoms of T cell lymphopenia.
RESULTS AND DISCUSSION
Animal care was in accordance with institutional guidelines. Map of the PAC clone used for pronuclear injection, genotyping of the offspring, and copy number of the Ian5 transgene are shown in Fig. 1. The BN PAC clone (∼150 kb) contained the normal allele of the Ian 5 gene and four simple sequence repeat markers used for subsequent genotyping of the offspring (Fig. 1A). The PAC clone was injected in circular form into the pronuclei of lymphopenic F344.lyp/lyp rats following a protocol reported earlier (15, 16). A female lymphopenia rescue transgenic (LRT) founder was detected (Fig. 1B, lane 5) from a total of 60 rats born from the injected eggs. This relatively low transgenic rate may be species dependent rather than dependent on the form of the injected DNA (3). When mated with F344.lyp/lyp males, this founder passed the transgenes to approximately half of its offspring. All four genetic markers (D4Rhw2, D4Rhw8, D4Rhw5, and D4Rhw4) that span the bulk of the PAC transgene (and flank the Ian5 gene, Fig. 1B, lane 2) showed BN allele sizes in the DNA from BN rats (lane 3), as well as in the DNA isolated from the transgenic (lanes 5, 6, 8, and 9) but not the F344.lyp/lyp rats (lanes 4 and 7), indicating that the entire molecule of the BN-derived PAC clone has been incorporated into the genome of the transgenic rats. The ratio of rIan5/rGAPDH PCR products (by quantitative real-time PCR) was 2.0 times higher in the LRT rats than in nontransgenic littermates, indicating that the transgenic rats have two extra copies of the rIan5 transgene from the PAC clone (Fig. 1C). All transgenics, when mated with nontransgenic F344.lyp/lyp partners, passed the transgenic DNA to approximately half of their progeny (45 of 83), indicating that the transgene was inherited in a Mendelian fashion. The lack of segregation indicates that both copies of Ian5 transgene were, most likely, integrated at a single site within the rat genome.
We compared the Ian5 mRNA expression in the thymus, mesenteric lymph nodes, spleen, and lungs of the LRT rats with that of nontransgenic littermates (i.e., F344.lyp/lyp), using quantitative real-time RT-PCR (Fig. 2A). The relative abundance of the transcript in the thymus, lymph nodes, and spleen of the transgenic rats was significantly higher (P < 0.05) than in the organs of lymphopenic F344.lyp/lyp subjects, indicating that transcription of transgenic Ian5 took place in the transgenic rats. Our previous study showed that in lymphoid organs of lymphopenic animals, the Ian5 transcript levels were reduced (13).
To determine whether the protein level was also corrected by the transgene, we compared Ian5 protein concentrations in thymus cell lysates isolated from the LRT transgenic rats to those isolated from the F344.lyp/lyp, F344.lyp/+, or BN and F344.+/+ rats, having 0, 1, or 2 copies of Ian5 gene, respectively (Fig. 2B). Western blots demonstrated a gene dose effect on Ian5 protein levels. Although totally undetectable in the lymphopenic F344.lyp/lyp strain, the protein expression in the F344.lyp/+, BN, and LRT transgenic rats was 56, 86, and 87%, respectively, of that observed in the nonlymphopenic F344.+/+ rats. The sequence of normal rIan5 predicts a protein of 35 kDa. The frame-shift mutation predicts a truncated protein of 11 kDa, in which the last 215 amino acids of wild-type Ian5 are replaced by 19 other amino acids (9, 13). Thus in the present experiment, the transgene from the BN rat produced the expected 35-kDa protein encoded by Ian5. The protein level in the transgenic animals was very similar to that observed in the BN rats, suggesting that the two normal alleles from the BN rats incorporated in the transgenic rats were translated normally and were controlled by BN genomic DNA. The slightly lower levels of the protein in this strain compared with the F344.+/+ rats may most likely reflect genetic difference between these two strains.
Next we compared the percentage of circulating T cells between the LRT transgenic, F344.lyp/lyp (including nontransgenic littermates), and Sprague-Dawley (SD) rats using flow cytometry after staining the α-β T cell receptor (TCR) with a monoclonal anti-rat antibody (R73) specific to the α-β TCR-positive T cell (Fig. 3). All of the nontransgenic littermates used for the T cell count were lymphopenic, demonstrating a low percent (14.0 ± 1.1%) of the α-β T cells. At the same time, none of the hemizygote transgenic rats was lymphopenic, having on average 48.8 ± 1.9% of the R73-positive T cells in the peripheral blood. The level of T cells in the LRT rats was similar to that observed in the nonlymphopenic SD or F344.+/+ (17) strain. We also compared the percentage of T cell subsets, CD4 and CD8, in spleen and mesenteric lymph nodes of nonlymphopenic F344.+/+, lymphopenic F344.lyp/lyp, and LRT rats. The levels of CD4 and CD8 cells in the LRT rats were significantly higher (P < 0.05) than those in the lymphopenic animals and comparable to the levels observed in the nonlymphopenic strain (data not shown).
Regarding the molecular mechanism underlying Ian5 function, recent reports indicate that the T cell lymphopenia in rats harboring a mutated Ian5 gene involves mitochondrial dysfunction leading to apoptosis of this cell population (8, 11, 19). In rat and human lymphocytes, Ian5 protein is probably anchored to the mitochondrial outer membrane by the hydrophobic domain at the COOH terminus (19, 25). Thus the mutated Ian5 cannot function because it is probably not correctly localized within the cellular organelle. Indeed, it was specifically shown that the lymphocytes expressing truncated Ian5 protein have decreased mitochondrial membrane potential and a defect in mitochondrial integrity leading to a substantially increased propensity to undergo spontaneous apoptosis (19). Furthermore, Ian5 has been shown to counteract radiation- or okadaic acid-induced apoptosis in human nonimmune cells, suggesting that its anti-apoptotic function may not be restricted to the immune system (23).
Thus transgenic incorporation of the normal allele of the Ian5 gene into the F344.lyp/lyp rat genome having a mutated Ian5 allele brought about elevated levels of transcript and protein together with normal levels of T cells in the peripheral blood and lymphoid organs, i.e., provided complete rescue of lymphopenia. For this reason, the present finding provides direct in vivo evidence that the single nucleotide mutation of the Ian5 gene is responsible for the lymphopenia in the F344.lyp/lyp rats.
In addition to the Ian5 gene, the transgenic PAC contained other genes as well, including Ian7, Ian1, Ian6, Ian3, Ian2, and Ian4 (13). On the basis of the results of our recombinant rat studies, we can fully exclude the Ian7, Ian1, Ian6, and Ian3 genes to contribute to the lymphopenia in this strain (13), whereas the Ian4 gene seems not to be expressed in the rat (E. A. Rutledge, unpublished observations). Regarding the Ian2 gene, we observed a single nucleotide polymorphism (T/C) between lymphopenic and nonlymphopenic BB rats. This polymorphism causes a predicted change in amino acid from methionine in nonlymphopenic to threonine in lymphopenic rats in the COOH-terminal region of the predicted protein. The BN Ian2 gene matches that of the nonlymphopenic BB rats. Since this variation occurs in the COOH-terminal end of the protein and at a position that is lacking functional recognition such as a predicted GTP binding domain, it is far less dramatic than the frame-shift mutation in the Ian5 gene and seems unlikely therefore to contribute to lymphopenia (E. A. Rutledge, unpublished observations).
Altogether, we consider it unlikely that another nearby gene without clear defect would rescue the lymphopenia phenotype while the correction of the Ian5 gene would have no obvious effect. The large size of the PAC clone used in this transgenic experiment (Fig. 1A) ensured copy number-dependent and integration site-independent expression levels, as expected (18). This most likely allowed for complete rescue of the lymphopenic phenotype.
Whereas previous studies demonstrated that the loss-of-function mutation in the Ian5 gene is associated with T cell lymphopenia, here we corrected the lymphopenia phenotype by an in vivo complementation approach using a PAC clone that harbors a full-length copy of the Ian5 gene in transgenic rats. The present transgenic rescue of T cell lymphopenia in a whole animal provides direct genetic evidence of the involvement of a novel Ian5 gene in the normal development of T cells. Furthermore, this work demonstrates that transgenic rescue in the rat is a practical and definitive method in revealing the function of a novel gene.
This study was supported by National Institutes of Health Grants AI-42380 (to A. Lernmark), HL-57921 (to M. Michalkiewicz), American Diabetes Association grant 1-02-JF-06 to R. A. Ettinger, and a mentor-based fellowship to A. Lernmark for D. H. Moralejo.
We thank Kriss M. Knestaut, Suzanne D. Lobaton, and Mae J. Racadio for technical support, and we thank Beverly Ventura for editing the manuscript.
C. D. Sigmund served as the review editor for this manuscript submitted by Editor H. Jacob.
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: M. Michalkiewicz, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, PO Box 26509, Milwaukee, WI 53226-0509 (E-mail:).
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