Development and characterization of Thy*IκBα-SI transgenic mice

We developed and characterized a new transgenic model where NF-κB activity is inhibited only in mature neurons. Transgenic mouse strain Thy*IκBα-SI was created using transdominant super inhibitor NF-κB (IκBα-SI), which is a multimutant form of IκBα inhibitory protein cloned into specifi c neutral Thy-1.2 cassette. Detailed molecular analysis showed that the transgene and its product (IκBα-SI protein) are expressed in the nervous system of transgenic mice. In situ hybridization showed that Thy*IκBα-SI in the nervous system is expressed exclusively in neurons. The developed model provides wide opportunities for studying functional role of NF-κB in mature neurons in the central and peripheral nervous system in vivo.     

Neuron-specific transgene expression of Bcl-XL but not Bcl-2 genes reduced lesion size after permanent middle cerebral artery occlusion in mice.

Protective effects after focal cerebral ischemia were assessed in transgenic mice that overexpress in a neuron-specific fashion mouse Bcl-XL or human Bcl-2. Both Bcl genes were under the control of the same mouse Thy-1 regulatory sequences resulting in very similar expression patterns in cortical neurons. Furthermore, these sequences direct lateonset (i.e. around birth) expression in brain, thus minimizing effects of transgene expression during brain development. Effects on infarct volume were measured using MRI after permanent occlusion of the middle cerebral artery (MCA). When compared to their non-transgenic littermates, Thy1mbcl-XL mice showed a significant 21% reduction in infarct size whereas Thy1hbcl-2 mice did not reveal any reduction. These findings suggest a selective protective advantage of Bcl-XL as compared with Bcl-2 in this mouse model for human stroke.     

Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic mice.

The use of the Cre-loxP recombination system allows the conditional inactivation of genes in mice. The availability of transgenic mice in which the Cre recombinase expression is highly cell type specific is a prerequisite to successfully use this system. We previously have characterized regulatory regions of the mouse flk-1 gene sufficient for endothelial cell-specific expression of the LacZ reporter gene in transgenic mice. These regions were fused to the Cre recombinase gene, and transgenic mouse lines were generated. In the resulting flk-1-Cre transgenic mice, specificity of Cre activity was determined by cross-breeding with the reporter mouse lines Rosa26R or CAG-CAT-LacZ. We examined double-transgenic mice at different stages of embryonic development (E9.5-E16.5) and organs of adult animals by LacZ staining. Strong endothelium-specific staining of most vascular beds was observed in embryos older than E11.5 in one or E13.5 in a second line. In addition, the neovasculature of experimental BFS-1 tumors expressed the transgene. These lines will be valuable for the conditional inactivation of floxed target genes in endothelial cells of the embryonic vascular system.     

ERT2-Cre诱导的Rosa26R-YFP报告基因系统标记小鼠造血干祖细胞的条件和效率检测

目的 探索ERT2-Cre诱导的黄色荧光蛋白（YFP）报告基因系统标记小鼠造血干祖细胞的条件和效率.方法 通过基因型分型的方法,从Rosa26R-YFP小鼠与ERT2-Cre小鼠杂交得到的后代中,筛选出YFP及ERT2-Cre双阳性转基因杂交小鼠;采用免疫磁珠法从双阳性转基因小鼠骨髓细胞中富集c-kit＋造血干祖细胞群进行体外培养,并用不同浓度4-羟基它莫西芬（OHT）作用不同时间以诱导YFP表达;流式细胞术检测c-kit＋造血干祖细胞中YFP的表达量.结果 Rosa26R-YFP小鼠与ERT2-Cre小鼠的杂交后代共9只,采用基因型分型方法筛选出双阳性转基因小鼠5只;体外培养的c-kit＋细胞部分形态发生变化,提示存在细胞分化;流式细胞术检测到c-kit＋细胞中YFP表达量可达13.7％.结论 YFP报告基因系统在OHT诱导的ERT2-Cre作用下,能有效标记小鼠造血干祖细胞.     

Age- and Gene-Dosage–Dependent Cre-Induced Abnormalities in the Retinal Pigment Epithelium

To conditionally inactivate genes in the retinal pigment epithelium (RPE) transgenic mouse strains have been developed, in which Cre recombinase (Cre) expression is driven by an RPE-specific gene promoter. The RPE is a quiescent epithelium, and continuous expression of Cre could affect its function. Here, we tested the hypothesis that continuous postnatal Cre expression in the RPE may lead to cellular abnormalities, which may depend on both age and Cre gene dosage. We therefore examined the eyes of homozygous and heterozygous VMD2-Cre mice at various ages. In VMD2-Cre heterozygous mice variable progressive age-dependent RPE abnormalities were noticed, including attenuation of phalloidin and cytoplasmic active β-catenin staining, reduced cell size, and loss of the typical honeycomb pattern of RPE morphology in those RPE cells that stained for Cre. These morphological RPE abnormalities were not noticed in Cre-negative RPE cells in VMD2-Cre or age-matched control mice. In addition, an abnormal number and morphology of cell nuclei were noticed in a subset of Cre-expressing RPE cells in aged heterozygous VMD2-Cre mice, whereas more severe nuclear abnormalities were observed already in young homozygous VMD2-Cre mice. Thus, continuous postnatal expression of Cre causes abnormalities in the RPE in an age- and Cre gene dosage-dependent manner, which needs to be considered in the interpretation of gene targeting studies in the RPE.The retinal pigment epithelium (RPE) is a nonregenerative single layer epithelium in the adult eye that is essential for the visual cycle and photoreceptor function.¹ Degenerative changes in the RPE affect retinal function and cause several ocular diseases. The fact that this epithelium does not continuously regenerate postnatally could make this epithelium more susceptible to accumulative genetic alterations that affect its overall function and morphology.Because of its critical role in vision and for the maintenance of photoreceptor function, understanding biological processes in the RPE are fundamentally important. Conditional gene inactivation studies have been undertaken to determine RPE-specific functions of certain genes. Several transgenic mouse strains have been developed that express Cre recombinase (Cre) in the RPE driven by a gene promoter that is active in this epithelium. These mice allow targeted inactivation of a gene in the RPE when crossed with mice that carry floxed alleles for the gene of interest.^2–7 Most RPE-specific Cre strains that have been reported show an incomplete Cre expression pattern in the RPE, with patches of Cre-positive RPE cells adjacent to Cre-negative RPE cells. Thus, it is important to correlate phenotypic changes in the RPE with the Cre expression pattern. Furthermore, the extent of Cre expression is variable, and it has been reported that mice of the same transgenic strain show a high degree of variability of Cre expression, with the observation of complete silencing of Cre expression in some mice that carry the Cre allele.² Cre-mediated RPE toxicity has been reported in Trp1-Cre transgenic mice,^3,8 raising the question whether this is a strain-specific observation or whether Cre expression in the RPE results in toxicity as a general phenomenon that may depend on the level and duration of cellular Cre expression in the RPE. The Trp1-Cre strain shows RPE-specific Cre expression already at embryonic day 10.5, which suggests that RPE-specific abnormalities in these mice may result from developmental abnormalities due to Cre expression at the onset of RPE morphogenesis. Whether Cre expression in the RPE postnatally would affect its viability and function is not known.In several other cell types Cre-mediated cell toxicity has been reported, with degenerative changes that correlated to the degree of Cre expression.^9–11 This Cre toxicity may result from genetic alterations due to sequences in the genome that may be targeted by Cre. Cre catalyzes recombination between two loxP recognition sites, which is characterized by a short recognition sequence.^12,13 Reports have shown that Cre expression can lead to DNA cleavage in mammalian cells at sites with homology to the loxP recognition sites (pseudo-loxP sites), which results in genetic alterations in mammalian cells that can significantly affect cell function.^14,15It is likely that the RPE is particularly susceptible to Cre-mediated toxicity, because it is a nonregenerative epithelium that would accumulate genetic changes with progressive age. To test this hypothesis, VMD2-Cre mice (also named BEST1-Cre mice) that express Cre in the RPE after postnatal day 10 were examined for Cre-mediated RPE abnormalities.² If Cre expression by itself leads to phenotypic changes in the RPE, we hypothesized that these changes would increase with progressive age and the amount of Cre expression in the RPE. Indeed, we show that RPE abnormalities in VMD2-Cre mice progressed with age and were more pronounced in mice that were homozygous for the Cre allele than in heterozygous mice. These findings have important implications for conditional inactivation studies in the RPE, and they suggest that the RPE is susceptible to Cre-mediated cellular dysfunction. Phenotypes due to RPE-specific conditional inactivation of a target gene need to be interpreted in the context of Cre-mediated cellular abnormalities, and in all studies mice heterozygous for Cre with age-matched heterozygous Cre control mice should be used. The conclusions that can be made in RPE-specific gene inactivation studies that use these mice, and likely with other RPE-specific Cre strains as well, need to carefully consider the observed variability of RPE abnormalities even among Cre heterozygous mice.     

Evidence for baseline retinal pigment epithelium pathology in the Trp1-Cre mouse

The increasing popularity of the Cre/loxP recombination system has led to the generation of numerous transgenic mouse lines in which Cre recombinase is expressed under the control of organ- or cell-specific promoters. Alterations in retinal pigment epithelium (RPE), a multifunctional cell monolayer that separates the retinal photoreceptors from the choroid, are prevalent in the pathogenesis of a number of ocular disorders, including age-related macular degeneration. To date, six transgenic mouse lines have been developed that target Cre to the RPE under the control of various gene promoters. However, multiple lines of evidence indicate that high levels of Cre expression can be toxic to mammalian cells. In this study, we report that in the Trp1-Cre mouse, a commonly used transgenic Cre strain for RPE gene function studies, Cre recombinase expression alone leads to RPE dysfunction and concomitant disorganization of RPE layer morphology, large areas of RPE atrophy, retinal photoreceptor dysfunction, and microglial cell activation in the affected areas. The phenotype described herein is similar to previously published reports of conditional gene knockouts that used the Trp1-Cre mouse, suggesting that Cre toxicity alone could account for some of the reported phenotypes and highlighting the importance of the inclusion of Cre-expressing mice as controls in conditional gene targeting studies.     

Possible role of cytotoxic T cells in acute liver injury in hepatitis C virus cDNA transgenic mice mediated by Cre/loxP system

A line of hepatitis C virus (HCV) transgenic mice was established previously that was mediated by Cre/loxP system using HCV cDNA, including core, E1, E2 and NS2 genes. Intravenous infection of a recombinant adenovirus that expresses Cre DNA recombinase (AxCANCre) induced HCV structural protein expression in the liver of transgenic mice. HCV core protein production and transgene recombination in the mouse liver were serially evaluated after AxCANCre infusion. Core proteins were expressed efficiently and transgene was almost completely recombined in the liver of mice after 3 days and then the levels of both core protein production and transgene recombination decreased continuously for 28 days. However, 30.6% of the transgene recombination remained at 28 days and only 2.7% of core production remained at 28 days after infection. Compared with nontransgenic controls, the serum alanine aminotransferase levels in transgenic mice were significantly higher 10, 14, and 21 days after adenovirus infection. Histological scoring also indicated severe pathological changes in the liver of transgenic mice after adenovirus infection. AxCANCre infusion increased CD8+ lymphocyte infiltration into the liver of transgenic mice compared with that of non-transgenic controls. Furthermore, cytotoxic T lymphocytes (CTLs) isolated from transgenic mice during liver injury were specific for the HCV proteins. These results suggest that HCV structural proteins expressed in the liver of transgenic mice enhanced liver injury. HCV-specific CTLs may be to enhance hepatitis. Thus, the present HCV transgenic mouse model provides a useful model of liver injury due to HCV, and the host immune response may play a pivotal role(s) in the pathogenesis of HCV.     

A hCXCR1 transgenic mouse model containing a conditional color-switching system for imaging of hCXCL8/IL-8 functions in vivo

To address the functions of human CXCL8 (hCXCL8)/IL-8 through hCXCR1 in vivo, we have developed a humanized, transgenic mouse for hCXCR1. This mouse line is versatile and allows for a variety of functional analyses using bioimaging, including Cre/loxP-mediated, tissue-specific hCXCR1 expression in a spatiotemporal manner; a color-switching mechanism, which uses spectrum-complementary, genetically encoded green and red fluorescence markers to label the hCXCR1-expressing cells [enhanced GFP (eGFP)] against the background [monomeric red fluorescent protein (mRFP)]; a bioluminescent marker, which is present in the hCXCR1-expressing cells; and an exogenous cell surface marker (eGFP moiety) in the hCXCR1-expressing cells, which facilitates identification, isolation, and targeting of these cells. The established, transgenic founder line RCLG3A (TG(+)) expresses only mRFP and does so ubiquitously. When the RCLG3A mice are crossed with the tamoxifen-inducible, whole-tissue Cre mice (ROSA26-Cre/Esr(+/-)), administration of tamoxifen induces whole-body hCXCR1 expression and color-switching. When RCLG3A mice are crossed with thymocyte-specific Cre mice (Lck-Cre(+/+)), the hCXCR1 expression and color-switching are restricted in a lineage-specific manner. This mouse line can be used to understand the functions of hCXCL-8 in vivo. In addition, our approach and vectors can be used to establish other tissue-specific, transgenic mice in conjunction with multifunctional cell markers, which facilitate cell imaging, tracing, and manipulation in vivo.     

Cre-mediated site-specific recombination in zebrafish embryos.

Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.     

实时荧光定量PCR检测转基因小鼠外源基因拷贝数方法的建立和应用

目的建立实时荧光定量PCR方法检测CaMKⅡα-Cre转基因小鼠外源基因拷贝数的方法。方法以CaMKⅡα-Cre转基因首建鼠及其仔代阳性鼠为研究对象,利用绝对定量的实时荧光定量PCR法检测转基因小鼠的拷贝数,并筛选出纯合子小鼠再经遗传育种方法以确定为纯合子小鼠。结果绝对定量标准曲线公式为:△Ct=-2.402log5N(拷贝数)+8.654,相关系数为0.9999,扩增效率为95.4%。三只CaMKⅡα-Cre首建鼠的拷贝数分别为19、7、5;三个转基因小鼠品系均成功获得纯合子小鼠。结论成功建立了检测转基因小鼠外源基因拷贝数的实时荧光定量PCR方法,该方法可用于检测各种转基因小鼠中外源基因的拷贝数。     

心外膜细胞标记基因介导的Cre重组酶在双转基因小鼠心脏的表达

目的:检测心外膜细胞标记基因Tbx18介导的Cre重组酶在双转基因小鼠心脏的表达及分布。方法:将Tbx18-Cre小鼠与ROSA26报告小鼠交配,收获不同怀孕天数(12.5天、13.5天、14.5天)的胚胎,用PCR技术筛选双转基因阳性胚胎,取阳性胚胎心脏进行冰冻切片、X-Gal染色。结果:PCR检测每只小鼠胚胎中约一半胚胎为双转基因阳性,这些胚胎心脏的心外膜、左心室壁及室间隔均可检测到X-Gal阳性的蓝色细胞;且随着胚胎心脏的发育,蓝色细胞显著增多。结论:心外膜细胞标记基因Tbx18介导的Cre重组酶能有效地在双转基因胚胎小鼠心脏表达,表明Tbx18在心脏发育过程中扮演重要角色。     

条件化转染MT基因小鼠模型的建立

目的建立四环素诱导调控的MT转基因小鼠模型。为血管瘤试验研究及MT致瘤机理研究奠定基础。方法通过PCR方法从鸡的基因组序列中克隆绝缘子元件；构建四环素诱导调控的条件化转基因质粒，并将其调控元件和受控元件串联成一个载体，在两元件之间插入绝缘子，以减轻两者之间的相互干扰。为提高转基因的表达效率，在转基因盒的上游亦插入绝缘子元件；将MT基因亚克隆至此载体；转基因载体功能行细胞瞬转试验及半定量逆转录-PCR验证后，将其在体外扩增、酶切、回收，行小鼠受精卵原核注射，获得转染MT基因阳性小鼠；强力霉素体外诱导部分阳性鼠MT基因表达，使其具有血管瘤表型。结果绝缘子元件成功克隆；成功构建条件化转基因载体；体外试验证实目的基因的表达受强力霉素的严格调控；通过原核注射，获得5只转基因阳性鼠；2只行体外诱导2月后。1只出现血管瘤表型，逆转录．PCR检测证实MT表达。其余3只阳性小鼠在传代建系中。结论条件化MT转基因小鼠模型成功构建，此小鼠模型能够为血管瘤的试验研究及MT致瘤机理的体内研究提供一定的基础。     

Natural killer cells target HCV core proteins during the innate immune response in HCV transgenic mice

The mechanism of the innate immune response to hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We used HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins regulated by the Cre/loxP switching expression system, to examine the innate immune response to HCV structural proteins. Twelve hours after HCV transgene expression, HCV core protein levels in Tg mouse livers were 15–47 pg/mg. In contrast, in Tg mice with a depletion of natural killer (NK) cells, we observed much higher levels of HCV core proteins (1,597 pg/ml). Cre‐mediated genomic DNA recombination efficiency in the HCV‐Tg mice was strongly observed in NK cell‐depleted mice between 0.5 and 1 day as compared to non‐treated mice. These data indicated that NK cells participate in the elimination of core‐expressing hepatocytes in the innate immune responses during the acute phase of HCV infection. J. Med. Virol. 82:1545–1553, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of hepatitis C virus replication by herbal extract: Phyllanthus amarus as potent natural source

We previously established inducible-hepatitis C virus (HCV) transgenic mice, which expressed the HCV gene (nucleotides 294-3435) encoding the core, E1, E2, and NS2 proteins. The expression of these proteins is regulated by the Cre/loxP system and an adenovirus vector (AdV) that expresses Cre DNA recombinase (Cre) controlled by the CAG promoter (AxCANCre). Recent studies have demonstrated that AxCANCre injection alone results in severe liver injury by induction of the adenovirus protein IX (Ad-pIX) gene. As a result, HCV protein expression in transgenic mice livers was only short-term. In contrast, the EF1α promoter-bearing AdV induces slight Ad-pIX gene expression without inducing severe liver injury. Therefore, in the present study, we developed a Cre-expressing AdV that bears the EF1α promoter (AxEFCre) to express HCV protein in the transgenic mouse livers. In the non-transgenic mice injected with AxCANCre, alanine aminotransferase (ALT) levels were elevated and severe liver inflammation occurred; this was not observed in AxEFCre-injected mice. In contrast, AxEFCre-injected HCV transgenic mice showed milder liver inflammatory responses that were clearly due to HCV protein expression. Moreover, the AxEFCre injection enabled the transgenic mice to persistently express HCV protein. These results indicate that use of AxEFCre efficiently promotes Cre-mediated DNA recombination in vivo without a severe hepatitis response to AdV. This inducible-HCV transgenic mouse model using AxEFCre should be useful for research on HCV pathogenesis.     

Neuron-specific activation of murine cytomegalovirus early gene e1 promoter in transgenic mice

The brain is the main target in congenital cytomegalovirus (CMV) infection and immunocompromised patients. No definite evidence that a CMV has special affinity for the central nervous system (CNS) has been published. Here, we generated transgenic mice with an e1 promoter/enhancer region connected to the reporter gene lacZ. Surprisingly, expression of the transgene was completely restricted to the CNS in all lines of transgenic mice. The transgene was expressed in subpopulation of neurons in the cerebral cortex, hippocampus, diencephalon, brainstem, cerebellum, and spinal cord in all of the lines. Non-neuronal cells in the CNS were negative for transgene expression. Activation of the transgene was first observed in neurons of mesencephalon in late gestation, and then the number of positive neurons increased in various parts of the brain as development proceeded. Upon infection of the transgenic mouse brains with MCMV, the location of the activated neurons became more extensive, and the number of such neurons increased. These results suggest that there are host factor(s) that directly activate the MCMV early gene promoter in neurons. This neuron-specific activation may be associated with persistent infection in the brain and may be responsible for the neuronal dysfunction and neuronal cell loss caused by CMV infection.     

Efficient ROSA26-Based Conditional and/or Inducible Transgenesis Using RMCE-Compatible F1 Hybrid Mouse Embryonic Stem Cells

The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state.     

应用接头PCR克隆慢病毒介导的转基因小鼠整合位点旁侧序列

目的 为鉴定慢病毒介导的转基因小鼠中外源基因的整合位点信息,应用接头PCR克隆整合位点旁侧序列.方法 小鼠基因组总DNA酶解后与设计的接头片段连接,根据慢病毒的LTR序列设计巢式PCR引物,克隆转基因小鼠整合位点旁侧序列.结果 成功克隆到转基因小鼠整合位点的旁侧序列,经过测序定位于小鼠染色体上.结论 作为反向PCR的改进,本方法可用于转基因小鼠整合位点旁侧序列的克隆, 为分析整合位点与外源基因表达之间的关系等提供了科学依据.     

Production and characterization of cytotoxic Thy-1 antibody-secreting hybrid cell lines. Detection of T cell subsets.

Responses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells. For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7D 5, which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons. Serologically, F7D 5 Thy-1.2 antibody was found to behave as a conventional Thy 1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1 producing lines are available for research purposes.