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PubMed2005

Only low levels of exogenous N-acetyltransferase can be achieved in transgenic mice.

Therapeutic and environmental aromatic amines and hydrazines are substrates for the arylamine N-acetyltransferases (NAT). In all, 10 transgenic lines containing either the human NAT1 or NAT2 transgene were developed using multiple promoters. The presence of the transgene was confirmed by determining copy number, mRNA and enzyme activity. Despite some lines having high copy numbers of the transgene, only modest or no increases in enzymatic activity could be found in a variety of tissues. The NAT1 transgene could not be bred to homozygosity. The cytomegalovirus (CMV)-promoted NAT1 transgene increased endogenous Nat1 mRNA levels in liver and had little effect on endogenous Nat2 mRNA levels. The presence of the CMV-promoted NAT2 transgene appeared to suppress endogenous hepatic Nat2 mRNA, but did not alter Nat1 mRNA levels. The failure to achieve high expression of any of the transgenes suggests that overexpression of NAT genes may have harmful effects during development.

Animals↗
PubMed2000

Glomerulosclerosis and viral gene expression in HIV-transgenic mice: role of nef.

BACKGROUND: Human immunodeficiency virus (HIV)-associated nephropathy is characterized by focal segmental glomerulosclerosis and microcystic tubular dilation. We have previously described a mouse transgenic for a Deltagag-pol HIV-1 genome, which develops glomerulosclerosis, cutaneous papillomas, and cataracts. METHODS: We developed mice transgenic for a Deltagag-pol-nef HIV genome in order to investigate the role of the nef gene in these phenotypes. RESULTS: One transgenic line, X5, expressed HIV mRNA in kidney and consistently manifested focal segmental glomerulosclerosis and tubular dilation by six weeks of age. Northern analysis indicated that renal transgene expression was higher in the Deltagag-pol-nef mice compared with the Deltagag-pol mice. In situ hybridization and immunostaining demonstrated HIV RNA and protein expression within the glomerular epithelial cells and tubular epithelial cells. These cell types showed histologic evidence of toxicity, including vacuolation and detachment from basement membrane, and exhibited increased rates of apoptosis. These data suggest that the renal disease seen in the Deltagag-pol-nef transgenic mouse may be caused by the expression of HIV genes within renal epithelial cells, that this expression may induce cellular toxicity, including apoptosis, and that nef is not required for the induction of renal disease. We have previously described mice bearing the nef gene, which do not manifest renal disease. In further experiments, Deltagag-pol-nef mice were bred with nef mice; these dual-transgenic mice developed renal disease that generally resembled that seen in Deltagag-pol-nef mice, but with somewhat more severe glomerulosclerosis and less severe tubulointerstitial injury. RESULTS: The results of these transgenic studies suggest that the role of nef is complex and may act both to reduce transgene expression and to potentiate glomerular injury induced by other HIV-1 gene products.

AIDS-Associated Nephropathy↗
PubMed2001

Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus.

Transgenic mice expressing MyrAkt from a proximal Lck promoter construct develop thymomas at an early age, whereas transgenic mice expressing constitutively active Lck-AktE40K develop primarily tumors of the peripheral lymphoid organs later in life. The thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in size but contains fewer, larger cells than the thymus of nontransgenic control and AktE40K transgenic mice. Earlier studies had shown that cell size and cell cycle are coordinately regulated. On the basis of this finding, and our observations that the oncogenic potential of Akt correlates with its effect on cell size, we hypothesized that mechanisms aimed at maintaining the size of the thymus dissociate cell size and cell cycle regulation by blocking MyrAkt-promoted G(1) progression and that failure of these mechanisms may promote cell proliferation resulting in an enlarged neoplastic thymus. To address this hypothesis, we examined the cell cycle distribution of freshly isolated and cultured thymocytes from transgenic and nontransgenic control mice. The results showed that although neither transgene alters cell cycle distribution in situ, the MyrAkt transgene promotes G(1) progression in culture. Freshly isolated MyrAkt thymocytes express high levels of cyclins D2 and E and cdk4 but lower than normal levels of cyclin D3 and cdk2. Cultured thymocytes from MyrAkt transgenic mice, on the other hand, express high levels of cyclin D3, suggesting that the hypothesized organ size control mechanisms may down-regulate the expression of this molecule. Primary tumor cells, similar to MyrAkt thymocytes in culture, express high levels of cyclin D3. These findings support the hypothesis that tumor induction is caused by the failure of organ size control mechanisms to down-regulate cyclin D3 and to block MyrAkt-promoted G(1) progression.

Animals↗
PubMed2000

Interleukin-5-mediated allergic airway inflammation inhibits the human surfactant protein C promoter in transgenic mice.

Allergen challenge in the lung of humans and animals is associated with surfactant dysfunction, but the mechanism of this effect has not been established. By using a murine model of asthma we now report the effect of allergen-induced airway inflammation on the expression of transgenes regulated by the human surfactant protein (hSP)-C promoter. The hSP-C 3.7-kilobase pair promoter was used to direct the expression of eotaxin, an eosinophil-selective chemokine, into the lungs of several transgenic lines. As expected, the transgenic mice expressed increased amounts of eotaxin mRNA and protein compared with wild-type mice. Surprisingly, following allergen challenge, there was a marked down-regulation of transgene mRNA in three independent transgenic lines. The down-regulation was in contrast to other related proteins such as endogenous eotaxin and surfactant protein D levels, which were both increased following allergen challenge. Consistent with specific down-regulation of the eotaxin transgene, there was no increase in pulmonary eosinophil levels in the transgenic mice above that found in wild-type mice. Analysis of hSP-C transgenic mice with distinct reporter genes and 3'-untranslated regions revealed that allergen challenge was directly affecting the hSP-C promoter. We hypothesized that allergen-induced down-regulation of the hSP-C promoter was related to the eosinophilic inflammation. To test this, we blocked eosinophilic inflammation in the lungs by treating mice with neutralizing antiserum against interleukin-5. Interestingly, this treatment also blocked allergen-induced inhibition of the hSP-C promoter. These results establish that allergic airway inflammation is associated with up-regulation of the surfactant proteins primarily involved in immunity, whereas down-regulation of the surfactant protein primarily involved in maintaining airway patency. Furthermore, the marked down-regulation of the hSP-C promoter is interleukin-5-dependent, implying a critical role for eosinophilic inflammation. These results suggest that alterations in surfactant protein levels may contribute to immune and airway dysfunction in asthma.

3' Untranslated Regions↗
PubMed1998

LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs.

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.

Animals↗
PubMed1998

Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene.

The random insertion of transgenes into the genomic DNA of mice usually leads to widely variable levels of expression in individual founder lines. To study the mechanisms that cause variegation, we designed a transgene that we expected to variegate, which consisted of a beta-globin locus control region 5' HS-2 linked in tandem to a tagged human beta-globin gene (into which a Lac-Z cassette had been inserted). All tested founder lines exhibited red blood cell-specific expression, but levels of expression varied >1000-fold from the lowest to the highest expressing line. Most of the variation in levels of expression appeared to reflect differences in the percentage of cells in the peripheral blood that expressed the transgene, which ranged from 0.3% in the lowest expressing line to 88% in the highest; the level of transgene expression per cell varied no more than 10-fold from the lowest to the highest expressing line. These differences in expression levels could not be explained by the location of transgene integration, by an effect of beta-galactosidase on red blood cell survival, by the half life of the beta-galactosidase enzyme or by the age of the animals. The progeny of all early erythroid progenitors (BFU-E colony-forming cells) exhibited the same propensity to variegate in methylcellulose-based cultures, suggesting that the decision to variegate occurs after the BFU-E stage of erythroid differentiation. Collectively, these data suggest that variegation in levels of transgene expression are due to local, integration site-dependent phenomena that alter the probability that a transgene will be expressed in an appropriate cell; however, these local effects have a minimal impact on the transgene's activity in the cells that initiate transcription.

Age Factors↗
PubMed2000

Pre-selection of integration sites imparts repeatable transgene expression.

Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for expression of a randomly integrated HPRT marker lying 5' to an Oct4/ lacZ transgene. 794 clones were assessed in vitro for appropriate down-regulation of lacZ following differentiation. Two clones were chosen for further analysis which displayed appropriate and inappropriate gene regulation (clones 710 and 91, respectively). Three developmental promoters (thyroglobulin, Hox2.6 and Myf5) were then sequentially introduced into the original insertion sites in each clone (710 and 91) by homologous recombination, to drive expression of lacZ. Transgenic embryos were assessed for their ability to direct lacZ expression to tissues in which the respective promoter sequences are normally active. The site which appropriately down-regulated lacZ in vitro (710) also showed appropriate in vivo regulation of lacZ from the three developmental promoters. Site 91, however, directed an additional pattern of ectopic expression, which was common to all four promoters. Pre-selection of genomic sites for the introduction of transgenes by gene targeting improves the repeatability of transgene expression and provides an efficient means of single copy transgene introduction by homologous recombination.

Animals↗
PubMed1997

A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate.

Age Factors↗
PubMed2004

A gammaGT-AT1A receptor transgene protects renal cortical structure in AT1 receptor-deficient mice.

To understand the physiological role of angiotensin type 1 (AT(1)) receptors in the proximal tubule of the kidney, we generated a transgenic mouse line in which the major murine AT(1) receptor isoform, AT(1A), was expressed under the control of the P1 portion of the gamma-glutamyl transpeptidase (gammaGT) promoter. In transgenic mice, this promoter has been shown to confer cell-specific expression in epithelial cells of the renal proximal tubule. To avoid random integration of multiple copies of the transgene, we used gene targeting to produce mice with a single-copy transgene insertion at the hypoxanthine phosphoribosyl transferase (Hprt) locus on the X chromosome. The physiological effects of the gammaGT-AT(1A) transgene were examined on a wild-type background and in mice with targeted disruption of one or both of the murine AT(1) receptor genes (Agtr1a and Agtr1b). On all three backgrounds, gammaGT-AT(1A) transgenic mice were healthy and viable. On the wild-type background, the presence of the transgene did not affect development, blood pressure, or kidney structure. Despite relatively low levels of expression in the proximal tubule, the transgene blunted the increase in renin expression typically seen in AT(1)-deficient mice and partially rescued the kidney phenotype associated with Agtr1a(-/-)Agtr1b(-/-) mice, significantly reducing cortical cyst formation by more than threefold. However, these low levels of cell-specific expression of AT(1) receptors in the renal proximal tubule did not increase the low blood pressures or abolish sodium sensitivity, which are characteristic of AT(1) receptor-deficient mice. Although our studies do not clearly identify a role for AT(1) receptors in the proximal tubules of the kidney in blood pressure homeostasis, they support a major role for these receptors in modulating renin expression and in maintaining structural integrity of the renal cortex.

Animals↗
PubMed1996

Transgenic remodeling of the contractile apparatus in the mammalian heart.

The structure-function relationships of the sarcomeric proteins in the mammalian cardiac compartment remain ill-defined because of the lack of a suitable model in which they can be readily manipulated or exchanged in vivo. To establish the validity of the transgenic paradigm for remodeling the mammalian heart, the murine alpha -cardiac myosin heavy chain gene promoter was used to express a ventricular myosin light chain-2 transgene (MLC2v) in both the atria and ventricles of the adult animal. Expression resulted in high levels of the transgene's transcript in both compartments. In the ventricle, the transgene was expressed against the background expression of the normal isoform. In the atrium, the transgene's expression would be ectopic, in that normally, MLC2v expression is restricted to the ventricle. Ectopic expression of the transgene in the atria resulted in a complete replacement of the atrial myosin light chain-2 protein isoform, although the endogenous isoform's steady state transcript levels were unchanged. In contrast, ventricular expression of the transgene had no effect at the protein level, despite an eightfold increase in MLC2v transcript levels. The data show that sarcomeric protein stoichiometry is maintained rigorously via posttransciptional regulation and that protein replacement can be achieved through a single transgenic manipulation.

Animals↗
PubMed2004

Chick delta1-crystallin enhancer influences mouse alphaA-crystallin promoter activity in transgenic mice.

PURPOSE: Both the -366/+43 and the -282/+43 mouse alphaA-crystallin (or alphaA) promoters have been effective at driving transgene expression in lens fiber cells, but not in lens epithelium. Because the chick delta1-crystallin gene is expressed in lens epithelial cells, an enhancer was borrowed from this gene and linked to the alphaA promoter. This heterogenic enhancer/promoter construct was tested in transgenic mice to see whether it was active in both lens epithelium and fiber cells while retaining lens specificity. METHODS: The third intron of the chick delta1-crystallin gene, which contains a lens enhancer element, was added to the 5' end of the mouse alphaA promoter. We refer to this chimeric regulatory element as the deltaenalphaA promoter. To test its activity, we inserted coding sequences for five different genes. Transgenic mice were generated by pronuclear microinjection. Transgene expression patterns were analyzed by either X-gal staining, in situ hybridization or immunohistochemical staining. RESULTS: When deltaenalphaA-lacZ transgenic embryos were stained with X-gal at embryonic day (E)11.5, beta-galactosidase activity was detected only in the eye. Histologic sections of the stained embryos revealed that lacZ was expressed exclusively in the lens, in both epithelial and fiber cells. Transgenic mice were also generated using either the original alphaA- or the new deltaenalphaA promoter linked to an insulin cDNA. In situ hybridizations confirmed that the short alphaA promoter targeted prenatal insulin expression specifically to the lens fiber cells, whereas the deltaenalphaA promoter was active in both lens epithelial and fiber cells. Developmental studies of the deltaenalphaA-insulin mice showed that the deltaenalphaA promoter became active at the lens pit stage and remained active in all lens cells, even at postnatal ages. The deltaenalphaA promoter also successfully directed expression of SV40 T-antigen (TAg), human E2F2, and dominant negative Sprouty2 (dn-Spry2) genes to lens epithelial and fiber cells. The lens specificity of the deltaenalphaA promoter was maintained in minigenes with different types of introns and polyadenylation signals. CONCLUSIONS: A new lens-specific regulatory element was generated-the deltaenalphaA promoter, which can drive high levels of transgene expression in both lens epithelium and fiber cells throughout development. This modified promoter can be used for future transgenic studies of signal transduction and cell cycle regulation in lens epithelial cells.

Animals↗
PubMed2006

A transgenic mouse model for human autosomal dominant cataract.

PURPOSE: To characterize lenses from transgenic mice designed to express mutant and wild-type alphaA-crystallin subunits. METHODS: A series of transgenic mouse strains was created to express mutant (R116C) and wild-type human alphaA-crystallin in fiber cells of the lens. Dissected lenses were phenotypically scored for the presence and extent of opacities, fiber cell morphology, and posterior suture morphology. Gene transcripts derived from integrated transgenes were detected by reverse transcriptase-PCR. Distribution of expressed transgenic protein was determined by immunohistochemical staining of lens tissue sections. The abundance of endogenous and transgenic lens proteins was estimated by quantitative Western blot analysis. RESULTS: Expression of R116C mutant alphaA-crystallin subunits resulted in posterior cortical cataracts and abnormalities associated with the posterior suture. The severity of lens abnormalities did not increase between the ages of 9 and 30 weeks. With respect to opacities and morphologic abnormalities, lenses from transgenic mice that express wild-type human alphaA-crystallin subunits were indistinguishable from age-matched nontransgenic control mice. Similar phenotypes were observed in different independent lines of R116C transgenic mice that differed by at least two orders of magnitude in the expression level of the mutant transgenic protein. CONCLUSIONS: The results show that lens opacities and posterior sutural defects occur when mutant R116C alphaA-crystallin subunits are expressed on the background of wild-type endogenous mouse alpha-crystallins. Low levels of R116C alphaA-crystallin subunits are sufficient to induce lens opacities and sutural defects.

Animals↗
PubMed1999

Expression and regulation of human sex hormone-binding globulin transgenes in mice during development.

Human sex hormone-binding globulin (SHBG) is produced by hepatocytes and transports sex steroids in the blood. The rat gene encoding SHBG is expressed transiently in the liver during fetal life, but it is not expressed in the liver postnatally, and the small amounts of SHBG in rat blood are derived from gonadal sources. To study the biosynthesis and function of human SHBG in an in vivo context, we have produced several lines of transgenic mice that contain either 11 kb (shbg11) or 4.3 kb (shbg4) portions of the human shbg locus. The expression and regulation of these transgenes have now been studied during fetal and postnatal development. In situ hybridization of an shbg11 transgenic mouse fetus at 17.5 days postcoitus located human shbg transcripts only in duodenal epithelial cells and hepatocytes. Temporal differences in the hepatic expression of mouse shbg and human shbg transgenes during late fetal development were reflected in corresponding differences in mouse and human SHBG levels in fetal and neonatal mouse blood. Serum concentrations of human SHBG increased during the first weeks of life regardless of gender until about 20 days of age in shbg11 mice, but after this time they continued to increase only in the males. This sexual dimorphism was reflected in corresponding differences in human SHBG messenger RNA (mRNA) abundance in the livers of these animals. However, it was not observed in shbg4 mice, in which hepatic production of plasma SHBG continued to increase after puberty regardless of gender. Serum testosterone and SHBG levels correlated in all sexually mature shbg transgenic mice. Human shbg transcripts were detectable only in testes of shbg11 mice and increased progressively in abundance from 10 days of age until the animal reached sexual maturity at 30 days of age, with appreciable increases occurring well before any changes in serum testosterone concentration. In the kidney, SHBG mRNA levels accumulated earlier in shbg11 than in shbg4 mice, and the expression of both types of transgenes was sexually dimorphic, with much higher SHBG mRNA levels in the kidneys of male mice. As increases in SHBG mRNA in the male kidneys coincided with increases in serum testosterone during sexual maturation, we reasoned that shbg transgene expression is androgen dependent in the kidney. This was confirmed by demonstrating that a decrease in SHBG mRNA abundance in male mouse kidneys after castration could be reversed by 5alpha-dihydrotestosterone treatment. Moreover, exogenous androgen increased human SHBG mRNA levels in the kidneys of female mice. In summary, comparisons of how different human shbg transgenes are expressed in vivo provides information about the positions of potential regulatory sequences that may control the hormonal regulation and tissue-specific expression of this gene during development.

Aging↗
PubMed2000

Transgenic analysis of the response of the rat calbindin-D 9k gene to vitamin D.

The promoter of the calbindin-D 9k (CaBP9k) gene, previously analyzed in transgenic mice, contains all of the information necessary for expression of a transgene similar to the endogenous gene and also for an appropriate response to vitamin D. In the present study we first investigated the role of a putative vitamin D-responsive element (9k/VDRE), located at nucleotides -489 to -445 on the rat CaBP9k promoter gene, using transgenic mice. As expected, the pattern of transgene expression in mice carrying this putative VDRE mutated in its whole promoter context was similar to that in mice bearing the wild-type sequence. These transgenic mice also responded to 1,25-dihydroxyvitamin D3 in the same way as those bearing the wild-type transgene and as those carrying a transgene with a large deletion (from -2894 to -117) eliminating the putative 9k/VDRE. Thus, the putative 9k/VDRE is not required for the control of rat CaBP9k gene expression by vitamin D in vivo. We also found that responsiveness to 1,25-dihydroxyvitamin D3 depends on the site at which the transgene is integrated into the host genome, in a tissue-specific manner. These data together with the fact that vitamin D-responsive sequences are present in a two-module region (from -3731 to -2894 and/or -117 to +365) and that this region does not contain any classical VDRE show that the CaBP9k gene is submitted to a non-conventional control by vitamin D.

Animals↗
PubMed2004

Transgenic mice overexpressing both amyloid beta-protein and perlecan in pancreatic acinar cells.

Heparan sulfate proteoglycans such as perlecan are thought to facilitate amyloid fibril formation. Tg3695 mice overexpress perlecan core protein in many tissues including the brain and pancreas. Tg13592 mice overexpress the signal plus 99-amino acid carboxyl terminal sequences (C99) of amyloid beta-protein precursor in multiple tissues and develop amyloid deposits in the pancreas. To investigate a role of perlecan in beta-amyloidosis, we established doubly transgenic mice by crossing the two lines of transgenic mice. The expression levels of the two transgenes remained unchanged in the brain and pancreas and the doubly transgenic mice did not develop amyloid deposits in the brain up to 19-months of age. Amyloid load detected by thioflavine S in the pancreas of the doubly transgenic mice was not significantly different from that in the transgenic littermates expressing only C99. Amyloid load in the pancreas increased during aging. We found a positive correlation between the Abeta-immunoreactive (non-fibrillar and fibrillar) and thioflavine S-positive (fibrillar) Abeta deposits in the single (C99) but not doubly transgenic mice. Our results suggest that perlecan does not independently influence amyloid formation in the pancreas of the transgenic mice and that there may be other factors that may modulate amyloid formation together with perlecan.

Aging↗
PubMed2006

Generation and characterization of a transgenic mouse model for pancreatic cancer.

AIM: To generate a SV40Tag transgenic tumor animal model and to study the mechanism underlying tumorigenesis. METHODS: A mammary gland expression vector containing SV40Tag DNA was generated. Transgene fragments were microinjeted into fertilized eggs of FVB mice. The genetically manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. PCR and Northern blot analysis were used for genotype analysis of F1 and F2 mice. Transgene expression was detected by RT-PCR and immunohistochemistry. RESULTS: SV40Tag gene was detected in two lines of transgenic mice. One of them delivered the transgene to F1 and a tumor was found in the pancreas of these mice. RT-PCR and immunohistochemistry showed that SV40Tag gene was expressed in the tumor. Pathological characterization of the transgenic mice demonstrated that the tumor belonged to pancreatic cystic neoplasm. CONCLUSION: SV40Tag transgenic mouse model can be successfully established. The transgenic mice develop a pancreatic tumor, which can be used for investigation of the molecular mechanism of tumorigenesis in vivo.

Animals↗
PubMed2000

Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice.

To assess the role of lysozyme in pulmonary host defense in vivo, transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated. Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar (BAL) lavage fluid was increased 2- or 4-fold relative to that in WT mice. Lung structure and cellular composition of BAL were not altered by the expression of lysozyme. Lysozyme activity in BAL was significantly increased (6.6- and 17-fold) in 5-wk-old animals from each transgenic line. To determine whether killing of bacteria was enhanced by expression of rat lysozyme, 5-wk-old transgenic mice and WT littermates were infected with 10(6) CFU of group B streptococci or 10(7) CFU of a mucoid strain of Pseudomonas aeruginosa by intratracheal injection. Killing of group B streptococci was significantly enhanced (2- and 3-fold) in the mouse transgenic lines at 6 h postinfection and was accompanied by a decrease in systemic dissemination of pathogen. Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines (5- and 30-fold). Twenty-four hours after administration of Pseudomonas aeruginosa, all transgenic mice survived, whereas 20% of the WT mice died. Increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo, and was associated with decreased systemic dissemination of pathogen and increased survival following infection.

Adjuvants, Immunologic↗
PubMed2000

Development of myelin oligodendrocyte glycoprotein autoreactive transgenic B lymphocytes: receptor editing in vivo after encounter of a self-antigen distinct from myelin oligodendrocyte glycoprotein.

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain kappa (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THkappa(mog)) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THkappa(mog) double-transgenic mice, the transgenic kappa(mog) L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2(-) mice, differentiation of THkappa(mog) B cells is blocked at an immature stage (defined by the B220(low)IgM(low)IgD(-) phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THkappa(mog) mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THkappa(mog) B cell receptor and induces editing of the transgenic kappa(mog) L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.

Animals↗