Chicken Beta-actin Research Articles

Generation of mice with conditionally activated transforming growth factor beta signaling through the TβRI/ALK5 receptor

We generated a transgenic mouse strain (LSL-TbetaRI(CA)) containing a latent constitutively active TGFbeta type I receptor (TbetaRI/ALK5) by using a knock-in strategy into the X chromosome-linked hypoxanthine phosphoribosyl-transferase (Hprt) locus. Transgene expression, under the control of the ubiquitous CAG (human cytomegalovirus enhancer and chicken beta-actin) promoter, is repressed by a floxed transcriptional "Stop" (LSL, Lox-Stop-Lox). In the presence of cre-recombinase, the "Stop" is excised to allow TbetaRI(CA) transgene expression. We showed that restricted expression of TbetaRI(CA) in T lymphocytes efficiently activates TGFbeta signaling and rescues the T-cell autoimmune disorders of TGFbetaRII conditional knockouts. Unexpectedly, our study reveals that TGFbeta signaling upregulation controls T-cell activation but does not impair their development or their peripheral homeostasis. In addition to the information provided on TGFbeta effects on T-cell biology, LSL-TbetaRI(CA) mouse constitutes an attractive tool to address the effect of TGFbeta signaling upregulation in any cell type expressing the cre-recombinase.

A small regulatory element from chromosome 19 enhances liver-specific gene expression

Tissue-specific promoters for gene therapy are typically too big for adeno-associated virus (AAV) vectors; thus, the exploration of small effective non-viral regulatory elements is of particular interest. Wild-type AAV can specifically integrate into a region on human chromosome 19 termed AAVS1. Earlier work has determined that a 347 bp fragment (Chr19) of AAVS1 has promoter and transcriptional enhancer activities. In this study, we further characterized this genetic regulation and investigated its application to AAV gene therapy in vitro and in vivo. The Chr19 347 bp fragment was dissected into three regulatory elements in human embryonic kidney cells: (i) TATA-independent promoter activity distributed throughout the fragment regardless of orientation, (ii) an orientation-dependent insulator function near the 5' end and (iii) a 107 bp enhancer region near the 3' end. The small enhancer region, coupled to the mini-CMV promoter, was used to drive the expression of several reporters following transduction by AAV2. In vivo data demonstrated enhanced transgene expression from the Chr19-mini-CMV promoter cassette after tail vein injection primarily in the liver at levels comparable to the chicken beta-actin promoter and higher than the liver-specific TTR promoter (>2-fold). However, we did not observe this increase after muscle injection, suggesting tissue-specific enhancement. All of the results support identification of a small DNA fragment (347 bp) from AAV Chr19 integration site capable of providing efficient and enhanced liver-specific transcription when used in recombinant AAV vectors.

Systemic overexpression of growth hormone (GH) in transgenic FVB/N inbred mice: an optimized model for holistic studies of molecular mechanisms underlying GH-induced kidney pathology

Transgenic mice overexpressing growth hormone (GH) display a plethora of phenotypic alterations and provide unique models for studying and influencing consequences of chronic GH excess. Since the first report on GH transgenic mice was published in 1982, many different mouse models overexpressing GH from various species at different levels and with different tissue specificities were established, most of them on random-bred or hybrid genetic background. We have generated a new transgenic mouse model on FVB/N inbred background, expressing bovine (b) GH under the control of the chicken beta-actin promoter (cbetaa). cbetaa-bGH transgenic mice exhibit ubiquitous expression of bGH mRNA and protein and circulating bGH levels in the range of several microg/ml, resulting in markedly stimulated growth and the characteristic spectrum of pathological lesions which were described in previous GH overexpressing mouse models. Importantly, a consistent sequence of renal alterations is observed, mimicking progressive kidney disease in human patients. The novel, genetically standardized GH transgenic mouse model is ideal for holistic transcriptome and proteome studies aiming at the identification of the molecular mechanisms underlying GH-induced pathological alterations especially in the kidney. Moreover, genetically defined cbetaa-bGH mice facilitate random mutagenesis screens for modifier genes which influence the effects of chronic GH excess and associated pathological lesions.

Improved Retinal Transduction In Vivo and Photoreceptor-specific Transgene Expression Using Adenovirus Vectors With Modified Penton Base

Adenovirus (Ad) vectors can be injected into human ocular tissues without producing adverse events and are therefore a promising means of gene transfer to the retina. However, when administered subretinally, Ad vectors primarily transduce the retinal pigment epithelium (RPE), whereas the majority of mutant gene products that cause photoreceptor (PR) degeneration are expressed exclusively in the PR cells. While it has been shown previously that pseudotyping of Ad can partially overcome the limited PR transduction by Ad5, we found that pseudotyping of Ad is not necessary for transduction of PR cells. We determined that, in the context of Ad, the cytomegalovirus (CMV) promoter is not significantly active in PRs. We compared expression levels from CMV and chicken beta actin (CBA) promoters in neural retina and found that CBA has a 173-fold greater potency than CMV. We also investigated the nature of the Ad-RPE interaction in murine retina and determined that the RGD domain in Ad penton plays a key role in RPE tropism. Deletion of the RGD domain coupled with use of the CBA promoter permitted transgene expression in neural retina approximately 667 times more efficiently than with Ad5 vectors. The use of these vectors in combination with a 4.7 kilobase (kb) rhodopsin promoter enabled transgene expression exclusively in PR cells in vivo.

Functional Analysis of Various Promoters in Lentiviral Vectors at Different Stages of In Vitro Differentiation of Mouse Embryonic Stem Cells

Given the therapeutic potential offered by embryonic stem (ES) cells, it is critical to optimize stable gene delivery and expression at different developmental stages of ES cell differentiation. Here, we systematically analyzed lentiviral vectors containing the following promoters: the human elongation factor 1alpha (EF1alpha) promoter, the human cytomegalovirus (CMV) immediate early region enhancer-promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), the human phosphoglycerate kinase 1 (PGK) promoter, the murine stem cell virus (MSCV) long terminal repeat (LTR), or the gibbon ape leukemia virus (GALV) LTR. Our results show that the EF1alpha promoter directed robust transgene expression at every stage of mouse ES cell differentiation, whereas the CMV promoter drove transgene expression only during late stages. Similarly, the CAG and PGK promoters drove transgene expression at a significant level only during late stages. The MSCV LTR and the GALV LTR exhibited much lower promoter activities at all stages. Interestingly, mouse ES cells transduced with the EF1alpha promoter-containing lentiviral vector lost most of their transgene expression during in vitro differentiation to neural precursors and neuronal cells. Our results demonstrate that different cellular and viral promoters exhibit very distinct and dynamic properties not only in terms of promoter strength but also with respect to differentiation stage-specific activity.

Cloning of pig serine proteinase inhibitor 9 and its use in protecting against apoptosis

The activity of granzyme B, a main effector molecule of natural killer (NK) cells and cytotoxic T lymphocytes, is regulated by the intracellular serine proteinase inhibitor 9 (PI-9). Pig PI-9 was first cloned, and the sequences that encode pig PI-9, including the start codon and stop codon, were identified. The cDNA was inserted into the cloning site of pCXN2 (chicken beta actin promoter and cytomegalovirus enhancer), transfected into pig endothelial cells (PEC), and several stable PEC clones were established. An NK cell-mediated cytolysis test was next applied to the PEC clones, using YT cells (an NK-like cell line). The PEC transfectants with pig PI-9 had a significant inhibitory effect on NK cell-mediated PEC lysis. The overexpression of the anti-apoptotic molecule, pig PI-9, has the potential for use in protecting graft cells from human NK cells.

Timing and Uniformity of Embryonic Gene Activation Affect Subsequent Pre-implantation Development of Cloned Bovine Embryos

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.

Cross-Species Function of the Pig C1 Esterase Inhibitor

Background The use of a bioartificial liver with pig cells for the treatment of fulminant hepatic failure will require research on the plasma complement regulatory proteins of the pig, because the liver produces most of the complement components and plasma complement regulatory proteins. In our previous study, the pig C1 esterase inhibitor (C1-INH), which functions as an inhibitor of the complement reaction in the first step of the classical pathway in the fluid phase, was cloned and some relevant features of the molecule were characterized, especially its cross-species regulation, in comparison with human C1-INH. In a further analysis, the species specificity of C1-INH was examined, using pig endothelial cells (PEC) and several types of sera. Materials and Methods The cDNA of pig C1-INH was used to produce the membrane type pC1-INH, pC1-INH-PI, and inserted into the cloning site of pCXN2 (chicken beta actin promoter). The pCX/pCl-INH-PI plasmid was then transfected into PEC to establish stable PEC with pCl-INH-PI. The expression of the pCl-INH-PI was evaluated by a FACS analysis, and complement-dependent cell lysis with human, dog, rabbit, and mouse sera was then assessed. Results The transfectant with pig Cl-INH-PI showed a high level of expression on PEC. The PEC transfectants showed an inhibitory effect on complement-dependent PEC lysis. Pig Cl-INH did not show the same suppressive effect for each serum. However, considering the alternative pathway activation of each serum on the pig cell membrane, it can be concluded that pCl-INH has a relatively small species restriction. Conclusion Pig Cl-INH, having a similar structure to human Cl-INH, shows a strong complement regulatory function on other species sera.

Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia.

Neuroglobin (Ngb), a protein related to myoglobin and hemoglobin but expressed predominantly in the brain, is induced by neuronal hypoxia and cerebral ischemia and protects against hypoxic or ischemic neuronal injury. We engineered transgenic mice that overexpress murine Ngb under the control of a chicken beta-actin promoter, resulting in enhanced Ngb expression in multiple cell types and multiple tissues, including brain and heart. In Ngb-overexpressing transgenic mice compared with wild-type littermates, the volume of cerebral infarcts after occlusion of the middle cerebral artery was reduced by approximately 30%, and the volume of myocardial infarcts produced by occlusion of the left anterior descending coronary artery was reduced by approximately 25%. Ngb overexpression was associated with enhanced expression of endothelial nitric oxide synthase in vascular endothelial cells. These findings extend prior evidence for cytoprotection by Ngb and suggest both direct (parenchymatous) and indirect (vasomotor) protective mechanisms.

Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 Over Expression in Transgenic Mice Suppresses Intestinal Neoplasia

The nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was identified as a proapoptotic, antitumorigenic protein in vitro, induced by many antitumorigenic and chemopreventive drugs including cyclooxygenase inhibitors. However, its antitumorigenic activity has not been elucidated in vivo. Transgenic mice were generated that ubiquitously overexpress human NAG-1 under the control of a chicken beta-actin promoter (CAG). The NAG-1 transgenic mice (NAG-(Tg+)) were characterized, and then the antitumorigenic activity was evaluated with 2 colorectal carcinogenesis models: chemical induction with azoxymethane and genetic induction using the Apc(Min+) mutation. NAG-(Tg+) showed no apparent phenotype other than a reduction in body weight, particularly in males. To examine whether NAG-1 expression would suppress intestinal tumorigenesis, the NAG-(Tg+) mice were treated with the colorectal carcinogen azoxymethane. NAG-(Tg+) mice developed 50% fewer aberrant crypt foci and no tumors, in comparison with nontransgenic littermates. This result demonstrates that expression of this human protein in vivo can suppress chemically induced carcinogenesis in the colon. The NAG-(Tg+) mice were also crossed with Apc(Min+) mice to determine the effect of the transgene on intestinal polyp formation. NAG-(Tg+) mice heterozygous for the Apc(Min+) mutation had a significantly reduced polyp load (60%) compared with nontransgenic Apc(Min+) littermates. Our results support NAG-1 as an important regulator of intestinal adenoma growth in vivo and suggest that NAG-1 may act as a tumor suppressor gene.

Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice

Previous studies have demonstrated a potential role of brain endogenous microglia and meningeal macrophages in inflammation and brain injury during bacterial meningitis. However, the contribution of previously engrafted monocytes and microglia to this process is still unknown. We therefore used genetically labelled bone marrow-derived cells from transgenic mice expressing the green fluorescent protein (GFP) under the chicken beta-actin promoter to deliver fluorescently labelled monocytes to the diseased brain. Approximately 24 hours after Streptococcus pneumoniae infection, GFP-expressing parenchymal microglia changed their morphology to an activated phenotype and upregulated major histocompatibility complex class II molecules. Bacterial meningitis increased the engraftment of GFP+ monocytes and their differentiation to microglia during the post-inflammatory period, but not during acute meningitis. Importantly, these newly recruited monocytes became an integral part of the pool of parenchymal microglia and contributed to the clearance of damaged tissue by increased lysosomal activity and close location to apoptotic cells. Thus, circulating cells entering the brain such as monocytes/macrophages might provide a potential cellular target for the treatment of the tissue damage following meningitis via peripheral cell therapy.

1032. rAAV Mediated Knock-Down of Tyrosine Hydroxylase in the Substantia Nigra Using a pol II Transcribed siRNA

The use of small interfering RNA (siRNA) has become increasingly popular in recent years for gene knockdown both in vivo and in vitro. To date, a majority of expression systems are utilizing a RNA pol III promoter. The pol III promoter, although supporting high levels of siRNA expression, cannot be easily regulated nor engineered to confer tissue specificity. In this study we utilize recombinant adeno associated virus (rAAV) to express siRNA targeting tyrosine hydroxylase (TH), a protein involved in the production of dopamine, using an expression cassette containing the chicken beta-actin (CBA) pol II promoter.

Investigation of the fate of Sry‐expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.

498. AAV Δ264CFTR Enhances Maturation of ΔF508CFTR and wt CFTR Expression

We have presented previously that high dose airway delivery of a pseudotyped AAV2/5 vector to the lungs of Rhesus macaques resulted in enhanced gene transfer and quantitative RNA expression as well as protein expression in non human primates without clinical toxicity. One major limitation to the success of AAV gene therapy for CF is that the large size of the CFTR cDNA insert fills the packaging capacity of AAV viral particles precluding inclusion of a highly efficient promoter. In order to overcome this limitation AAV2/5 pseudotyped vectors were generated expressing a truncated CFTR insert (|[Delta]|264) driven by a chicken beta actin promoter (rAAV-CB-|[Delta]|264-CFTR). We showed previously that this truncated form was capable of reconstituting physiologic function in vitro and in mouse models.

Effects of Heme Oxygenase-1 Transgenic Islets on Transplantation

Heme oxygenase-1 (HO-1) has been described as a protein capable of cytoprotection via radical scavenging and apoptosis prevention. The aim of this study was to analyze whether HO-1 overexpression in freshly isolated murine transgenic islets resulted in cell protection and improved in vivo functional performance after transplantation. We produced transgenic mice in which the human HO-1 transgene driven by chicken beta-actin promoter was expressed in the heart, liver, spleen, lung, kidney, muscle, intestine, and pancreas in Balb/c mice. One hundred fifty islets isolated from HO-1 transgenic and control Balb/c mice were syngeneically transplanted under the left kidney capsule of the streptozotocin-diabetic Balb/c mice. The recipients who underwent transplantation with HO-1 transgenic islets showed higher blood glucose than those with control islets at 4 weeks (320 ± 25 vs 189 ± 43 mg/dL; P < .05). Body weight was not significantly different between the 2 groups. Our data indicate transgenic islets with high HO-1 expression did not improve transplantation outcome.

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2S-M2 and a Tet repressor-KRAB fusion protein (tTSKRAB) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter Ptetbi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTSKRAB to Ptetbi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2S-M2 activates Ptetbi-1.

Localized Gene Expression Following Administration of Adeno-associated Viral Vectors via Pancreatic Ducts

Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting beta cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human alpha1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo, rAAV8) as well as different promoters (chicken beta-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 +/- 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 +/- 6 microg/ml). The chicken beta-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D.

Natural history of fetal cell microchimerism during and following murine pregnancy

In humans, fetal cells enter the maternal circulation during all pregnancies and can persist for decades. Human studies, however, are often limited by the number of subjects and the availability of healthy and diseased tissues for analysis. We sought to develop a murine model to establish the natural history of fetal cell microchimerism in various maternal tissues during and after healthy pregnancies resulting from congenic and allogenic matings. We bred C57BL/6J and DBA/2J virgin female mice to C57BL/6J males transgenic for the enhanced green fluorescent protein (GFP), which shows autosomal dominant inheritance with complete penetrance and is under the control of a ubiquitous chicken beta-actin promoter and a cytomegalovirus enhancer. During pregnancy and at different times after delivery, female mice were sacrificed. Tissues were collected and the presence of the gfp transgene and GFP+ cells was assessed by real-time quantitative PCR and by immunofluorescence. During pregnancy, microchimerism was detected in all tissues from mice carrying GFP+ fetuses. Fetal cells were often mononuclear. The frequency of fetal cells in the lungs was significantly higher compared to other tissues. The level of microchimerism was also significantly higher in congenic compared to allogenic matings. After delivery, the frequency of fetal cells decreased and fetal cells were undetectable at 2 and 3 weeks after the first delivery. However, some mice that had three gestations had detectable fetal cells 3 weeks after their last delivery. Using sensitive methods of detection, we demonstrate that fetal cell microchimerism occurs during all murine pregnancies. We describe a useful model for the study of the consequences of this phenomenon.

678. Inflammatory Cytokines Regulate Transgene Expression from Rheumatoid Synoviocytes Infected with Double Stranded Adeno-Associated Virus Vectors

A major drawback to current gene transfer strategies is the inability to regulate transgene expression. An ideal vector would provide local transgene expression at the site of the disease only when the disease is active. Recombinant adeno-associated virus (rAAV) appears to possess these important qualities. We monitored the effects of inflammatory cytokines on transgene expression in human rheumatoid arthritis fibroblast-like synoviocytes (FLS) infected with rAAV. Synovial cell cultures were infected with rAAV under control of the cytomegalovirus (CMV) promoter in the presence or absence of TNF|[alpha]|, IL-1|[beta]|, and/or IL-6. These cytokines were found to act synergistically to increase transgene expression in rAAV infected FLS up to 1000 fold. The enhanced expression required the continued presence of cytokines, although cytokine removal and re-addition demonstrated a remarkable ability of the cytokines to regulate transgene expression. The effect was most pronounced on cells infected with a double stranded version of AAV in which a rate-limiting step of viral transduction (second strand synthesis) is bypassed, and the effect was not promoter specific, as similar results were obtained using rAAV under control of the chicken beta actin promoter. mRNA levels of the transgene were demonstrated to increase up to 25-fold upon cytokine addition. The observed phenomenon was also phosphatidylinositol 3-kinase (PI3K) dependent, as addition of wortmannin reversibly inhibited cytokine-mediated increases in transgene expression. These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV-mediated gene transfer, making AAV an attractive gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis.

115. In Vivo Expression of Human Cob(I)alamin Adenosyltransferase Using rAAV Serotypes 1,2 and 8, an Approach to Gene Therapy for Methylmalonic Aciduria (MMA)

Top of pageAbstract Methylmalonic aciduria (MMA) is an autosomal recessive disease with an incidence of approximately 1 in 50,000 people in the United States. Symptoms include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000 fold greater then normal. Currently, eight different complementation groups (cblA-H) for MMA have been described. CblB MMA is caused by a deficiency in the enzyme Cob(I)alamin adenosyltransferase (ATR). ATR functions within the mitochondria matrix in the final conversion of inactive vitamin B12 to coenzyme B12, adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). AdoCbl and MCM catalyze methylmalonyl-CoA to succinyl-CoA, which is further metabolized in the TCA cycle. The human ATR cDNA was cloned into an rAAV vector containing the hybrid cytomegalovirus (CMV) enhancer with the chicken beta-actin promoter and AAV2-inverted terminal repeats and pseudotyped in AAV1, 2 or 8 capsids. AAV serotypes 2 and 8 were delivered by portal vein injection to six week old female C57/Bl6 mice at a low dose of 1|[times]|1010 particles and a high dose of 1|[times]|1011 particles. Eight-weeks post injection, these animals were sacrificed and their livers harvested. RNA, genomic DNA and protein were then extracted from these tissues and used for reverse transcriptase PCR, real time PCR, western blotting and ATR enzyme assays. Portions of the liver were also reserved for immunostaining. AAV1 and 2 were delivered intramuscularly to the tibialis anterior of C57/Bl6 at a low dose of 1|[times]|1010 and a high dose of 1|[times]|1011 particles. These animals were sacrificed at ten weeks and the injected muscles were harvested. RT-PCR was used to confirm transcript levels, and immunostaining was performed for tissue biodistribution. RESULTS: Using primer pairs specific to the CMV enhancer/chicken beta-actin promoter of rAAV vectors, genome copies/cell in the liver were found to 0.03, 2.03 and 0.10 for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Using primer pairs specific for the vector genome, a substantial increase of message was seen with AAV8 high dose over AAV2 dose. Similar mRNA expression was seen between the AAV8 low dose and AAV2 high dose animal tissues. Western blotting performed on mitochondrial protein extracts demonstrated the presence of pre-ATR and post-translocated protein in all three dose groups. Levels were comparable to control levels in the AAV8 low dose and AAV2 high dose animals. Levels three to five fold higher than control levels were observed in AAV8 high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the AAV8 high dose animals, compared to 9% and 5% transduction in AAV2 high dose and AAV8 low dose animals, respectively. In muscle, an increase of rAAV ATR message was seen in AAV1 high dose animal muscles as compared to AAV2 high dose animals.