BACKGROUND
alzheimer's disease  is a devastating neurodegenerative disorder that affects 7–10% of elderly individuals over  <dig> years of age and nearly 50% of those over  <dig> in the u.s  <cit> .
ad also can be inherited in an autosomal dominant manner, which causes a familial form of ad  that usually emerges at younger ages than does idiopathic ad  <cit> .
fad has been linked to mutations in the β-amyloid precursor protein   <cit> , as well as presenilin  <dig>  <cit>  and presenilin  <dig>  <cit> , which are critical components of the γ-secretase complex that liberates the amyloid-β peptide  from membranes.
aβ is the major proteinaceous component of senile plaques, and all known fad mutations increase the production of aβ or its tendency to aggregate.
two predominant forms of aβ, aβ <dig> and aβ <dig>  result from the proteolytic cleavage of app by β- and γ-secretases  <cit> .
because it is highly amyloidogenic, aβ <dig> is believed to play a particularly important role in the pathogenesis of ad  <cit> .
several transgenic  mouse models of ad have been generated to study the effects of app mutations.
the genetic background of mice has been shown to have a significant effect on plasma and brain aβ <dig> and aβ <dig> levels as well as on aβ-deposition in brain  <cit> .
outbred mouse lines show variability in aβ production and deposition, whereas aβ production is more consistent within an inbred transgenic line.
in this regard, the generation of transgenic rats on various genetic backgrounds  has been more difficult than mice and outbred rats   <cit> .
recently, outbred single-transgenic rats have been generated that express either human wild type  <cit>  or mutant app  <cit> .
transgenic wistar rats overexpressing human app <dig>  were used to investigate the role of app in the recovery process following cerebral ischemia  <cit> .
the authors report that despite the total app/aβ levels in cortex and hippocampus of happ <dig> rats being twice the level of non-transgenic controls, happ <dig> did not develop amyloid plaques with aging.
tg wistar rats overexpressing the appsw/ind transgene showed some intraneuronal aβ immunoreactivity but failed to develop β-amyloid deposits by  <dig> months of age  <cit> .
recently, folkesson et al.
<cit>  reported the development of an outbred sd rat line expressing appsw which begin to show extracellular aβ that is predominantly found in cerebrovascular blood vessels beginning at  <dig> months of age.
ruiz-opazo et al.
<cit>  reported generation of inbred appsw fischer- <dig> rats with a  <dig> % increase in app mrna expression in the brain of transgenic animals compared to non-transgenic cohorts at 12-months of age.
the appsw fischer- <dig> rats did not show evidence of extracellular amyloid deposits or senile plaques up to  <dig> months of age.
interestingly, appsw fischer- <dig> rats showed attenuated hippocampus-dependent learning and memory decline compared to non-transgenic cohorts as measured by morris water maze.
ruiz-opazo  <cit> and colleagues have postulated that within a particular expression range app or its derivatives my play a role in normal learning and memory, but when expressed at levels exceeding this threshold app and/or its metabolites could lead to neuronal loss and cognitive decline.
although mice have been extensively used in many areas of biomedical research, rat models have certain advantages over mice due to their larger size, unique genetics, and well-studied behavioral characteristics  <cit> .
rats are better suited for microsurgery, cell and tissue transplantation, in vivo functional analyses, and studies that require multiple sampling  <cit> .
in the context of neurological studies, stereotaxic injection is a commonly used method that is easier to perform with precision in rats than in mice.
for these reasons, it is advantageous to have germline tg rat models of neurodegenerative diseases like ad in order to better understand the underlying biochemical changes taking place during disease development and to test potential treatment options.
the ad rat models developed previously were generated mostly on outbred rat strains.
our aim was to develop an inbred app transgenic rat line with high app expression in the brain, as we believe this may be one of the important prerequisites for producing amyloid pathology in rat models of ad.
RESULTS
promoter characterization and selection
comparison of enhanced green fluorescent protein  expression driven from ubiquitin-c , cytomegalovirus , or platelet-derived growth factor  promoters in sd rat brains after stereotaxic injection of lentivirus showed that the ubi-c promoter yielded consistently superior egfp expression than did the cmv-egfp and pdgf-egfp viruses.
figure  <dig> shows egfp expression in rat brains three weeks post-injection of ubi-c-egfp and cmv-egfp lentiviruses.
enhanced gfp expression driven by cmv and pdgf promoters appeared to decrease dramatically over time.
in contrast, strong ubi-c- egfp expression persisted without apparent diminution at the longest time-point examined .
these results with ubi-c are consistent with previous findings using the ubi-c promoter in driving transgene expression  <cit> .
while stable, high-level transgene expression is highly desirable in tg animals, neuron-selective expression is also an important consideration in selecting an optimal promoter.
brain expression of the egfp transgene driven by a ubi-c promoter was examined in tg sd rats created by injection of egfp-lentivirus into fertilized zygotes.
strong egfp expression was seen in adult rats; by confocal microscopy, egfp expression appeared to be restricted to neurons .
when egfp distribution was directly compared to immunostaining with glial fibrillary acidic protein  to label astrocytes, there was strong divergence in the staining patterns.
to confirm the apparent restriction of egfp driven by the ubi-c promoter to neurons, primary cultures were established from e <dig> tg sd embryos and employed for colocalization studies using markers for neurons and glial cells.
confocal images of mixed primary cultures stained with neuron-specific beta tubulin and gfap is shown in figure 2j.
enhanced gfp is expressed in neurons, but not in glial cells, confirming the neuronal specificity of egfp expression driven by the ubi-c promoter.
transgenic founders
initial screening of app-transgenic founders and generation of homozygous transgenic rats
four pcr- and southern blot-positive appsw/ind tg rats were generated .
two of the founders were germline tg  and each had a single copy of the transgene.
the other two were not germline tg  and each contained two copies of the transgene.
the molecular weights of dna fragments containing the transgene that were obtained through bamhi digestion were  <dig> and  <dig> kb for app <dig> and app <dig> lines, respectively.
ecori digestion of genomic dna yielded  <dig>  and  <dig> kb fragments for app <dig> and app <dig> lines, respectively.
both app <dig> and app <dig> tg lines contained one copy of the transgene as determined by two separate restriction enzyme digestions .
different molecular weights of the transgenes in app <dig> and app <dig> animals is due to different transgene integration sites in these two transgenic lines.
both app <dig> and app <dig> lines were bred for five generations, demonstrating the stability of the transgene across generations; both lines appear normal and do not have any indication of unpredicted side effects of the transgenes.
app transgene expression
expression of the app transgene was determined by northern blot analysis in homozygous rats .
the app probe used for northern blot analysis hybridizes both the human app transgene as well as native rat app.
the blast score  of the  <dig> bp human app probe template to rat app mrna was  <dig>  the molecular weight of the app mrna was approximately  <dig>  kb.
northern blot analysis showed that the app <dig> line expressed the highest levels of app mrna, the app <dig> line expressed intermediate levels , and the native rat app mrna expression was the lowest.
expression of app showed a tissue-dependent variation.
the transgene was highly expressed in kidney and lung of both the app <dig> and app <dig> lines.
expression in brain was intermediate, and expression in liver and heart was the lowest for both tg lines .
similar expression patterns for rat app mrna were observed in non-transgenic rats, such that liver and heart contained lower app compared to brain, kidney and lung.
the average expression among the organs analyzed was  <dig>  times lower in wt rats than in app tg rats.
brain app expression in the app <dig> line was  <dig>  and  <dig>  times greater than in app <dig> and wt rats, respectively .
aβ elisa
serum levels of human aβ <dig> were determined in homozygous app <dig> , app <dig> , and hemizygous app <dig>  rats.
in addition,  <dig> rats from two tg parents, denoted as 'homozygosity status unknown', were included in the statistical analysis .
serum aβ <dig> was measurable in all app <dig> animals, and homozygous rats had  <dig>  times greater aβ <dig> than did hemizygous rats.
average serum aβ <dig> levels of the app <dig> line were  <dig> times lower than those of the app <dig> line.
in addition, aβ <dig> was undetectable in  <dig> out of  <dig> rats, which includes  <dig> homozygous rats.
serum levels of aβ <dig>  measured in two app <dig> homozygous rats, were  <dig> ±  <dig> pg/ml.
only one out of eleven app <dig> rats had measurable levels of aβ <dig>
brain immunohistochemistry
app transgene expression in c3- <dig> mice
human app cdna expression in tg c3- <dig> mice  <cit>  was analyzed to determine if a different promoter, the prion protein promoter , would also drive the expression of the human app cdna in a tissue-specific manner similar to that observed in app <dig> and app <dig> tg rats.
northern blotting results  showed that transgenic app expressed tissue-specifically in c3- <dig> mice as well.
the transgenic app expression was highest in brain, heart, kidney and lung, but very little app expression was detected in the liver of c3- <dig> mice.
relative expression in brain, kidney, liver and lung was similar in app <dig> and app <dig> rats and c3- <dig> mice.
however, in c3- <dig> mice, expression of app in heart was similar to app expression levels in brain, kidney and lung.
intriguingly, these two different tg species , in which the app cdna transgene is under the control of two unrelated promoters, show a similar pattern of tissue-specific expression
discussion
this study demonstrates the efficacy of generating inbred tg rats using a lentiviral vector, which is a novel approach for generating tg animals  <cit> .
despite dozens of tg mouse models of ad-like pathology, few tg rat lines are available for ad research  <cit> .
since the manifestation of specific genetic disorders in transgenic models is expected to be unique to each species and strain, it is essential to control phenotypic variations that stem from the genetic constitution of the background strain.
thus we chose the inbred fischer  <dig> rat strain for transgenic production in order to minimize individual variation among transgenic rats.
in transgenic mouse models of ad, lehman et al.
<cit>  reported that genetic background had a significant influence on the regulation of app and aβ deposition in tg mice that were created on different genetic backgrounds.
for our studies, we chose inbred fischer  <dig> rats due to their well-defined genetics as well as their common usage in studies involving aging  <cit> .
as fischer  <dig> rats age, their brains are increasingly susceptible to oxidative stress, which is known to correlate with many neurodegenerative diseases, including ad.
however, the approach that we employed to create germline tg appsw/ind lines using fischer  <dig> rats can be equally applied to other strains, providing unrestricted opportunities to create disease models on various genetic backgrounds.
in this paper, we report the successful generation of appsw/ind tg fischer  <dig> rats expressing human app <dig> containing the swedish and indiana mutations under the control of the ubi-c promoter using a lentiviral vector.
the app <dig> form was chosen because the predominant human app isoform expressed in neurons of the central nervous system  is the app <dig> isoform  <cit> .
additionally, we chose to construct the double-mutant app <dig> transgene to facilitate comparisons with existing app <dig> transgenic mouse and rat models, particularly the tg <dig> mice reported by hsiao et al.
<cit> .
long-lasting neuronal expression of transgenes is an important consideration in modeling neurodegenerative diseases such as ad.
our earlier efforts to create tg app-and ps1-overexpressing sd rats under the control of the cmv promoter using lentiviral vectors were successful in terms of efficient gene delivery.
however, the tg rats did not have the desired level of gene product as assessed by elisa and western blot analysis, even at  <dig> months of age .
this lack of expression may be attributable to the phenomenon of gene silencing, which has previously been observed with a number of promoters, including cmv.
in the present study, the easily detectable egfp reporter gene was used to compare egfp expression after stereotaxic injection of lentiviral vectors containing various promoters.
in these studies, cmv, pdgf, and ubi-c promoters all drove high-level expression of egfp in-vitro.
however, when tested in vivo after stereotaxic injection or creation of tg animals, cmv-egfp and pdgf-egfp expression decreased dramatically over time, whereas egfp expression remained strong for up to  <dig> months when driven by the ubi-c promoter.
characterization of transgene expression in rat brains revealed long-lasting and selective neuronal expression of genes driven by the ubi-c promoter.
stereotaxic injections were used to qualitatively screen cmv, ubic, and pdgf promoters for ability to drive egfp transgene expression within the hippocampus.
the choice of sd rats was based on the use of this strain in the stereotaxic mapping of the rat brain by paxinos & watson  <cit> .
the dentate gyrus injection coordinates extrapolated from the atlas have been experimentally verified in our laboratory using sd rats and therefore we used sd rats to examine egfp expression from the cmv, ubic, and pdgf promoters.
the goal of the promoter comparisons was not to characterize the expression patterns of the cmv, ubic, and pdgf promoter in the cns, which has previously been well documented, but rather to show qualitative examples of egfp expression within the hippocampus following focal injection of lentivirus.
stereotaxic injections were performed in dentate gyrus to allow qualitative assessment of gene expression within the hippocampal formation, a region particularly vulnerable to neuropathological insult in ad.
we determined that the egfp signal consistently appears to be both nuclear and cytoplasmic in vitro and in vivo, regardless of the method of transgene delivery or type of promoter.
similar results were obtained by wei et al.
<cit> , who showed that egfp diffused bidirectionally via the nuclear pore complex across the nuclear envelope.
to our knowledge, this is the first inbred, app-transgenic rat model of ad that has substantial quantities of aβ in serum.
prior to the generation of app <dig> and app <dig> transgenic rat strains, a fischer  <dig> inbred ad model expressing app  was reported by ruiz-opazo et al.
<cit> .
however, app expression in these tgappsw rats was only 56% greater than in wt rats.
in addition, app-transgenic, outbred wistar rats expressed  <dig>  times more app in hippocampus than did control rats  <cit>  in the current paper, we report  <dig>  times greater app expression in the brains of inbred fischer  <dig> rats than in wt controls.
due to the higher app expression, app <dig> rats could be useful models for examining the underlying mechanisms of ad progression and for developing and testing potential therapies for ad.
several mouse models have been generated to study the effects of app mutations.
hsia et al.
<cit>  generated tg appsw/ind mice.
the characterization of these tg mice indicated that the neurotoxic effects of aβ may not require plaque formation.
app <dig> mice express 7-fold more appsw than endogenous mouse app and develop aβ deposits at  <dig> months of age  <cit> .
tg app mice were generated using tissue-specific promoters such as enolase, platelet derived growth factor, and thy- <dig>  <cit> .
similarly, we show that promoter choice significantly influences the expression of the transgene, such that ubi-c is superior to cmv and pdgf in rats.
the present study describes the generation of app tg fischer  <dig> rats under the control of the ubi-c promoter, which drives transcription in all tissues relatively stably.
the expression of appsw/ind transgenic mrna was detectable in all tissues analyzed for both the app <dig> and app <dig> lines.
the expression of transgene was greater in the app <dig> line than in the app <dig> line.
since these tg rats were generated as models of ad-like pathology, the expression level of the transgene is particularly important.
the app <dig> line showed  <dig> times greater cerebral app expression compared to wt rats.
the expression of brain app in the app <dig> line was about half that of the app <dig> line.
in addition, serum aβ <dig> levels corroborate these findings.
the app <dig> line had significantly greater human aβ <dig> than did the app <dig> line.
in transgenic mouse models, the expression of app has reached levels as high as 10-fold greater than endogenous app  <cit> .
however, protein expression level is not the only determinant of ad phenotype, as lower expression of mutant forms of app can induce early and robust deposition of aβ in brain parenchyma and/or vasculature  <cit> .
while the anticipation of stable transgene expression was a key consideration in selecting the ubi-c promoter for our studies, selective neuronal expression driven by this promoter was unexpected.
our observations in brain and primary cultures from egfp-transgenic sd rats confirmed the highly preferential expression of egfp in neurons versus glial cells .
potentially even greater levels of selectivity are suggested by our characterization of appsw/ind expression in transgenic fischer  <dig> rats.
in these animals, human app was strongly expressed in neurons, but within the hippocampus, there was a strong demarcation based on the intensity of immunostaining in the pyramidal cell layer between ca <dig> and ca <dig> .
the basis for this selectivity is unclear, and additional studies will be required to fully evaluate the distribution of ubi-c promoter-driven transgene expression in brain.
we chose northern blot analysis to assess gene expression in app <dig> and app <dig> transgenic and wt rats and c3- <dig> mice.
this allowed us to confirm the size of the complete transcript in the transgenic animals.
samples collected from brain yield a higher band for app mrna and 18s rrna for both rat and mouse samples.
since both app mrna and 18s rrna migration was retarded in brain rna samples compared to the rest of the organs, we suspect that brain rna samples contain residual substances that impede the migration of brain rna.
these could be residual lipids, as the brain contains more fat compared to the other organs analyzed.
since the sequences of human and rat app are highly similar, the app probe used for northern blot analysis did not distinguish between human and rat app.
this enabled comparison of native rat app- and human app-transgene expression.
the transgene as well as the endogenous rat app gene expression patterns showed significant tissue-specificity.
interestingly, both the human app transgene and endogenous rat app mrna were more abundant in kidney and lung than in heart and liver.
although tissue-specific expression of app in humans was reported previously  <cit> , tissue-specific expression of the app transgene was unexpected in the transgenic rats because the ubi-c promoter drives expression of the human app transgene in this construct.
we have generated purinergic receptor y2-  transgenic rats using the same vector backbone, and expression of the p2ry <dig> transgene driven by the ubi-c promoter did not show tissue-specificity .
tissue specific expression of app transgene can be due to app mrna stability, rna silencing or transcription regulatory elements within app cdna.
the 3'utrs of human, rat and transgenic app are substantially different which reduces the possibility of tissue specific differences in app mrna stability.
in addition, we were unable to find a candidate sequence for rna silencing within the rat or human genome.
thus, regulation through enhancer or silencer elements within the app cdna is a possible explanation for tissue-specific expression regardless of the promoter that drives the expression of app.
a recent publication by collin and martens  <cit>  supports the presence of transcription regulatory role of app cdna.
in order to confirm tissue-specific expression of app, we analyzed app transgene expression in c3- <dig> transgenic mice, which is driven by the ubiquitously expressed prion protein promoter  <cit> .
expression levels of endogenous prion protein are similar in various tissues.
we suggest that the similarity of tissue-specific expression patterns of app transgenes that are driven by two different promoters  in two different species  strongly supports the presence of transcription regulatory elements within the app cdna.
the temporal and spatial expression differences in app-transgenic mice have been attributed to the promoters  used to drive expression  <cit> .
however, we believe that the changes in app expression patterns shown in our study cannot be explained by the promoter because of the stable expression driven by the ubi-c promoter in most tissues.
we hypothesize that elements within the cdna sequence may regulate the expression of app tissue-specifically.
identification of these elements might ultimately broaden treatment options for ad.
in conclusion, these app-transgenic rats could be a useful model in which to study the regulation of app expression as well as pathogenic mechanisms in ad.
CONCLUSIONS
this study shows the high efficiency of establishing stable, inbred germline transgenic rats by lentiviral gene delivery.
the app <dig> rats, which express high levels of human app, could be a valuable model of ad.
furthermore, the tissue-specific expression of the app transgene indicates the presence of regulatory elements within app cdna that could be a useful target for ad treatment or prevention.
our ongoing studies are aimed at intensive pathological and behavioral characterization of these unique app-transgenic rats.

 alzheimer's disease is a devastating neurodegenerative disorder that affects 7–10% of elderly individuals over years of age and nearly 50% of those over in the u.s. ad also can be inherited in an autosomal dominant manner, which causes a familial form of ad that usually emerges at younger ages than does idiopathic ad. fad has been linked to mutations in the β-amyloid precursor protein, as well as presenilin and presenilin, which are critical components of the γ-secretase complex that liberates the amyloid-β peptide from membranes. aβ is the major proteinaceous component of senile plaques, and all known fad mutations increase the production of aβ or its tendency to aggregate. two predominant forms of aβ, aβ and aβ result from the proteolytic cleavage of app by β- and γ-secretases. because it is highly amyloidogenic, aβ is believed to play a particularly important role in the pathogenesis of ad. several transgenic mouse models of ad have been generated to study the effects of app mutations. the genetic background of mice has been shown to have a significant effect on plasma and brain aβ and aβ levels as well as on aβ-deposition in brain. outbred mouse lines show variability in aβ production and deposition, whereas aβ production is more consistent within an inbred transgenic line. in this regard, the generation of transgenic rats on various genetic backgrounds has been more difficult than mice and outbred rats. recently, outbred single-transgenic rats have been generated that express either human wild type or mutant app. transgenic wistar rats overexpressing human app were used to investigate the role of app in the recovery process following cerebral ischemia. the authors report that despite the total app/aβ levels in cortex and hippocampus of happ rats being twice the level of non-transgenic controls, happ did not develop amyloid plaques with aging. tg wistar rats overexpressing the appsw/ind transgene showed some intraneuronal aβ immunoreactivity but failed to develop β-amyloid deposits by months of age. recently, folkesson et al. reported the development of an outbred sd rat line expressing appsw which begin to show extracellular aβ that is predominantly found in cerebrovascular blood vessels beginning at months of age. ruiz-opazo et al. reported generation of inbred appsw fischer- rats with a % increase in app mrna expression in the brain of transgenic animals compared to non-transgenic cohorts at 12-months of age. the appsw fischer- rats did not show evidence of extracellular amyloid deposits or senile plaques up to months of age. interestingly, appsw fischer- rats showed attenuated hippocampus-dependent learning and memory decline compared to non-transgenic cohorts as measured by morris water maze. ruiz-opazo and colleagues have postulated that within a particular expression range app or its derivatives my play a role in normal learning and memory, but when expressed at levels exceeding this threshold app and/or its metabolites could lead to neuronal loss and cognitive decline. although mice have been extensively used in many areas of biomedical research, rat models have certain advantages over mice due to their larger size, unique genetics, and well-studied behavioral characteristics. rats are better suited for microsurgery, cell and tissue transplantation, in vivo functional analyses, and studies that require multiple sampling. in the context of neurological studies, stereotaxic injection is a commonly used method that is easier to perform with precision in rats than in mice. for these reasons, it is advantageous to have germline tg rat models of neurodegenerative diseases like ad in order to better understand the underlying biochemical changes taking place during disease development and to test potential treatment options. the ad rat models developed previously were generated mostly on outbred rat strains. our aim was to develop an inbred app transgenic rat line with high app expression in the brain, as we believe this may be one of the important prerequisites for producing amyloid pathology in rat models of ad.  promoter characterization and selection comparison of enhanced green fluorescent protein expression driven from ubiquitin-c, cytomegalovirus, or platelet-derived growth factor promoters in sd rat brains after stereotaxic injection of lentivirus showed that the ubi-c promoter yielded consistently superior egfp expression than did the cmv-egfp and pdgf-egfp viruses. figure shows egfp expression in rat brains three weeks post-injection of ubi-c-egfp and cmv-egfp lentiviruses. enhanced gfp expression driven by cmv and pdgf promoters appeared to decrease dramatically over time. in contrast, strong ubi-c- egfp expression persisted without apparent diminution at the longest time-point examined. these results with ubi-c are consistent with previous findings using the ubi-c promoter in driving transgene expression. while stable, high-level transgene expression is highly desirable in tg animals, neuron-selective expression is also an important consideration in selecting an optimal promoter. brain expression of the egfp transgene driven by a ubi-c promoter was examined in tg sd rats created by injection of egfp-lentivirus into fertilized zygotes. strong egfp expression was seen in adult rats; by confocal microscopy, egfp expression appeared to be restricted to neurons. when egfp distribution was directly compared to immunostaining with glial fibrillary acidic protein to label astrocytes, there was strong divergence in the staining patterns. to confirm the apparent restriction of egfp driven by the ubi-c promoter to neurons, primary cultures were established from e tg sd embryos and employed for colocalization studies using markers for neurons and glial cells. confocal images of mixed primary cultures stained with neuron-specific beta tubulin and gfap is shown in figure 2j. enhanced gfp is expressed in neurons, but not in glial cells, confirming the neuronal specificity of egfp expression driven by the ubi-c promoter. transgenic founders initial screening of app-transgenic founders and generation of homozygous transgenic rats four pcr- and southern blot-positive appsw/ind tg rats were generated. two of the founders were germline tg and each had a single copy of the transgene. the other two were not germline tg and each contained two copies of the transgene. the molecular weights of dna fragments containing the transgene that were obtained through bamhi digestion were and kb for app and app lines, respectively. ecori digestion of genomic dna yielded and kb fragments for app and app lines, respectively. both app and app tg lines contained one copy of the transgene as determined by two separate restriction enzyme digestions. different molecular weights of the transgenes in app and app animals is due to different transgene integration sites in these two transgenic lines. both app and app lines were bred for five generations, demonstrating the stability of the transgene across generations; both lines appear normal and do not have any indication of unpredicted side effects of the transgenes. app transgene expression expression of the app transgene was determined by northern blot analysis in homozygous rats. the app probe used for northern blot analysis hybridizes both the human app transgene as well as native rat app. the blast score of the bp human app probe template to rat app mrna was the molecular weight of the app mrna was approximately kb. northern blot analysis showed that the app line expressed the highest levels of app mrna, the app line expressed intermediate levels, and the native rat app mrna expression was the lowest. expression of app showed a tissue-dependent variation. the transgene was highly expressed in kidney and lung of both the app and app lines. expression in brain was intermediate, and expression in liver and heart was the lowest for both tg lines. similar expression patterns for rat app mrna were observed in non-transgenic rats, such that liver and heart contained lower app compared to brain, kidney and lung. the average expression among the organs analyzed was times lower in wt rats than in app tg rats. brain app expression in the app line was and times greater than in app and wt rats, respectively. aβ elisa serum levels of human aβ were determined in homozygous app, app, and hemizygous app rats. in addition, rats from two tg parents, denoted as'homozygosity status unknown', were included in the statistical analysis. serum aβ was measurable in all app animals, and homozygous rats had times greater aβ than did hemizygous rats. average serum aβ levels of the app line were times lower than those of the app line. in addition, aβ was undetectable in out of rats, which includes homozygous rats. serum levels of aβ measured in two app homozygous rats, were ± pg/ml. only one out of eleven app rats had measurable levels of aβ brain immunohistochemistry app transgene expression in c3- mice human app cdna expression in tg c3- mice was analyzed to determine if a different promoter, the prion protein promoter, would also drive the expression of the human app cdna in a tissue-specific manner similar to that observed in app and app tg rats. northern blotting results showed that transgenic app expressed tissue-specifically in c3- mice as well. the transgenic app expression was highest in brain, heart, kidney and lung, but very little app expression was detected in the liver of c3- mice. relative expression in brain, kidney, liver and lung was similar in app and app rats and c3- mice. however, in c3- mice, expression of app in heart was similar to app expression levels in brain, kidney and lung. intriguingly, these two different tg species, in which the app cdna transgene is under the control of two unrelated promoters, show a similar pattern of tissue-specific expression discussion this study demonstrates the efficacy of generating inbred tg rats using a lentiviral vector, which is a novel approach for generating tg animals. despite dozens of tg mouse models of ad-like pathology, few tg rat lines are available for ad research. since the manifestation of specific genetic disorders in transgenic models is expected to be unique to each species and strain, it is essential to control phenotypic variations that stem from the genetic constitution of the background strain. thus we chose the inbred fischer rat strain for transgenic production in order to minimize individual variation among transgenic rats. in transgenic mouse models of ad, lehman et al. reported that genetic background had a significant influence on the regulation of app and aβ deposition in tg mice that were created on different genetic backgrounds. for our studies, we chose inbred fischer rats due to their well-defined genetics as well as their common usage in studies involving aging. as fischer rats age, their brains are increasingly susceptible to oxidative stress, which is known to correlate with many neurodegenerative diseases, including ad. however, the approach that we employed to create germline tg appsw/ind lines using fischer rats can be equally applied to other strains, providing unrestricted opportunities to create disease models on various genetic backgrounds. in this paper, we report the successful generation of appsw/ind tg fischer rats expressing human app containing the swedish and indiana mutations under the control of the ubi-c promoter using a lentiviral vector. the app form was chosen because the predominant human app isoform expressed in neurons of the central nervous system is the app isoform. additionally, we chose to construct the double-mutant app transgene to facilitate comparisons with existing app transgenic mouse and rat models, particularly the tg mice reported by hsiao et al.. long-lasting neuronal expression of transgenes is an important consideration in modeling neurodegenerative diseases such as ad. our earlier efforts to create tg app-and ps1-overexpressing sd rats under the control of the cmv promoter using lentiviral vectors were successful in terms of efficient gene delivery. however, the tg rats did not have the desired level of gene product as assessed by elisa and western blot analysis, even at months of age. this lack of expression may be attributable to the phenomenon of gene silencing, which has previously been observed with a number of promoters, including cmv. in the present study, the easily detectable egfp reporter gene was used to compare egfp expression after stereotaxic injection of lentiviral vectors containing various promoters. in these studies, cmv, pdgf, and ubi-c promoters all drove high-level expression of egfp in-vitro. however, when tested in vivo after stereotaxic injection or creation of tg animals, cmv-egfp and pdgf-egfp expression decreased dramatically over time, whereas egfp expression remained strong for up to months when driven by the ubi-c promoter. characterization of transgene expression in rat brains revealed long-lasting and selective neuronal expression of genes driven by the ubi-c promoter. stereotaxic injections were used to qualitatively screen cmv, ubic, and pdgf promoters for ability to drive egfp transgene expression within the hippocampus. the choice of sd rats was based on the use of this strain in the stereotaxic mapping of the rat brain by paxinos & watson. the dentate gyrus injection coordinates extrapolated from the atlas have been experimentally verified in our laboratory using sd rats and therefore we used sd rats to examine egfp expression from the cmv, ubic, and pdgf promoters. the goal of the promoter comparisons was not to characterize the expression patterns of the cmv, ubic, and pdgf promoter in the cns, which has previously been well documented, but rather to show qualitative examples of egfp expression within the hippocampus following focal injection of lentivirus. stereotaxic injections were performed in dentate gyrus to allow qualitative assessment of gene expression within the hippocampal formation, a region particularly vulnerable to neuropathological insult in ad. we determined that the egfp signal consistently appears to be both nuclear and cytoplasmic in vitro and in vivo, regardless of the method of transgene delivery or type of promoter. similar results were obtained by wei et al., who showed that egfp diffused bidirectionally via the nuclear pore complex across the nuclear envelope. to our knowledge, this is the first inbred, app-transgenic rat model of ad that has substantial quantities of aβ in serum. prior to the generation of app and app transgenic rat strains, a fischer inbred ad model expressing app was reported by ruiz-opazo et al.. however, app expression in these tgappsw rats was only 56% greater than in wt rats. in addition, app-transgenic, outbred wistar rats expressed times more app in hippocampus than did control rats in the current paper, we report times greater app expression in the brains of inbred fischer rats than in wt controls. due to the higher app expression, app rats could be useful models for examining the underlying mechanisms of ad progression and for developing and testing potential therapies for ad. several mouse models have been generated to study the effects of app mutations. hsia et al. generated tg appsw/ind mice. the characterization of these tg mice indicated that the neurotoxic effects of aβ may not require plaque formation. app mice express 7-fold more appsw than endogenous mouse app and develop aβ deposits at months of age. tg app mice were generated using tissue-specific promoters such as enolase, platelet derived growth factor, and thy-. similarly, we show that promoter choice significantly influences the expression of the transgene, such that ubi-c is superior to cmv and pdgf in rats. the present study describes the generation of app tg fischer rats under the control of the ubi-c promoter, which drives transcription in all tissues relatively stably. the expression of appsw/ind transgenic mrna was detectable in all tissues analyzed for both the app and app lines. the expression of transgene was greater in the app line than in the app line. since these tg rats were generated as models of ad-like pathology, the expression level of the transgene is particularly important. the app line showed times greater cerebral app expression compared to wt rats. the expression of brain app in the app line was about half that of the app line. in addition, serum aβ levels corroborate these findings. the app line had significantly greater human aβ than did the app line. in transgenic mouse models, the expression of app has reached levels as high as 10-fold greater than endogenous app. however, protein expression level is not the only determinant of ad phenotype, as lower expression of mutant forms of app can induce early and robust deposition of aβ in brain parenchyma and/or vasculature. while the anticipation of stable transgene expression was a key consideration in selecting the ubi-c promoter for our studies, selective neuronal expression driven by this promoter was unexpected. our observations in brain and primary cultures from egfp-transgenic sd rats confirmed the highly preferential expression of egfp in neurons versus glial cells. potentially even greater levels of selectivity are suggested by our characterization of appsw/ind expression in transgenic fischer rats. in these animals, human app was strongly expressed in neurons, but within the hippocampus, there was a strong demarcation based on the intensity of immunostaining in the pyramidal cell layer between ca and ca. the basis for this selectivity is unclear, and additional studies will be required to fully evaluate the distribution of ubi-c promoter-driven transgene expression in brain. we chose northern blot analysis to assess gene expression in app and app transgenic and wt rats and c3- mice. this allowed us to confirm the size of the complete transcript in the transgenic animals. samples collected from brain yield a higher band for app mrna and 18s rrna for both rat and mouse samples. since both app mrna and 18s rrna migration was retarded in brain rna samples compared to the rest of the organs, we suspect that brain rna samples contain residual substances that impede the migration of brain rna. these could be residual lipids, as the brain contains more fat compared to the other organs analyzed. since the sequences of human and rat app are highly similar, the app probe used for northern blot analysis did not distinguish between human and rat app. this enabled comparison of native rat app- and human app-transgene expression. the transgene as well as the endogenous rat app gene expression patterns showed significant tissue-specificity. interestingly, both the human app transgene and endogenous rat app mrna were more abundant in kidney and lung than in heart and liver. although tissue-specific expression of app in humans was reported previously, tissue-specific expression of the app transgene was unexpected in the transgenic rats because the ubi-c promoter drives expression of the human app transgene in this construct. we have generated purinergic receptor y2- transgenic rats using the same vector backbone, and expression of the p2ry transgene driven by the ubi-c promoter did not show tissue-specificity. tissue specific expression of app transgene can be due to app mrna stability, rna silencing or transcription regulatory elements within app cdna. the 3'utrs of human, rat and transgenic app are substantially different which reduces the possibility of tissue specific differences in app mrna stability. in addition, we were unable to find a candidate sequence for rna silencing within the rat or human genome. thus, regulation through enhancer or silencer elements within the app cdna is a possible explanation for tissue-specific expression regardless of the promoter that drives the expression of app. a recent publication by collin and martens supports the presence of transcription regulatory role of app cdna. in order to confirm tissue-specific expression of app, we analyzed app transgene expression in c3- transgenic mice, which is driven by the ubiquitously expressed prion protein promoter. expression levels of endogenous prion protein are similar in various tissues. we suggest that the similarity of tissue-specific expression patterns of app transgenes that are driven by two different promoters in two different species strongly supports the presence of transcription regulatory elements within the app cdna. the temporal and spatial expression differences in app-transgenic mice have been attributed to the promoters used to drive expression. however, we believe that the changes in app expression patterns shown in our study cannot be explained by the promoter because of the stable expression driven by the ubi-c promoter in most tissues. we hypothesize that elements within the cdna sequence may regulate the expression of app tissue-specifically. identification of these elements might ultimately broaden treatment options for ad. in conclusion, these app-transgenic rats could be a useful model in which to study the regulation of app expression as well as pathogenic mechanisms in ad.  this study shows the high efficiency of establishing stable, inbred germline transgenic rats by lentiviral gene delivery. the app rats, which express high levels of human app, could be a valuable model of ad. furthermore, the tissue-specific expression of the app transgene indicates the presence of regulatory elements within app cdna that could be a useful target for ad treatment or prevention. our ongoing studies are aimed at intensive pathological and behavioral characterization of these unique app-transgenic rats. 
in addition to idiopathic disease, ad is also associated with autosomal dominant inheritance, which causes a familial form of ad. alzheimer's disease is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. immunohistochemistry in brain showed that the human app transgene was expressed in neurons, but not in glial cells. northern blots showed that the human app transgene, driven by the ubiquitin-c promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. app and app rats expressed - and 3-times more app mrna, respectively, than did wild-type rats. the unexpected similarity in the tissue-specific expression patterns of endogenous rat app and transgenic human app mrnas suggests regulatory elements within the cdna sequence of app. fischer rats expressing human app driven by the ubiquitin-c promoter were generated via lentiviral vector infection of fischer zygotes. the app rat line expresses high levels of human app and could be a useful model for ad. tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of app gene regulation. determination of the elements that are responsible for tissue-specific expression of app may enable new treatment options for ad.
 alzheimer's disease is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. in addition to idiopathic disease, ad is also associated with autosomal dominant inheritance, which causes a familial form of ad. some instances of fad have been linked to mutations in the β-amyloid protein precursor. although there are numerous mouse ad models available, few rat ad models, which have several advantages over mice, have been generated.  fischer rats expressing human app driven by the ubiquitin-c promoter were generated via lentiviral vector infection of fischer zygotes. we generated two separate app-transgenic rat lines, app and app serum levels of human amyloid-beta were pg/ml for hemizygous and pg/ml for homozygous app animals. serum aβ levels in app homozygous rats were pg/ml. immunohistochemistry in brain showed that the human app transgene was expressed in neurons, but not in glial cells. these findings were consistent with independent examination of enhanced green fluorescent protein in the brains of egfp-transgenic rats. app and app rats expressed - and 3-times more app mrna, respectively, than did wild-type rats. northern blots showed that the human app transgene, driven by the ubiquitin-c promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. a similar expression pattern was also seen for the endogenous rat app. the unexpected similarity in the tissue-specific expression patterns of endogenous rat app and transgenic human app mrnas suggests regulatory elements within the cdna sequence of app.  this manuscript describes the generation of app-transgenic inbred fischer rats. these are the first human ad model rat lines generated by lentiviral infection. the app rat line expresses high levels of human app and could be a useful model for ad. tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of app gene regulation. determination of the elements that are responsible for tissue-specific expression of app may enable new treatment options for ad. 