eGFP Transgene Research Articles

9. Mobilization of Sendai Virus Vectors (SeVV) by Phylogenetic Related Human Pathogens: Implications for Future Vector Generations

In an attempt to make HIV-1 based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of the TATA box and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this 400bp U3 deletion are thought to have no LTR-directed transcription, and are thus called self-inactivating (SIN) lentivectors. As with wild-type LTR vectors, any transcripts initiated at the U3-R junction in the 5' LTR of SIN lentivectors contain the packaging signal found in the psi region and 5' portion of gag. Transcripts containing the packaging signal have the potential to be encapsidated and mobilized by wild-type HIV-1. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5' LTR which is responsible for the production of full-length vector genome transcripts. To verify that transcripts derived from the LTR in SIN lentivectors are competent for encapsidation and integration, we transduced a lentiviral packaging cell line with a standard SIN lentivector and then observed production of viable vector particles containing full-length SIN lentivector genomes. We also demonstrate that a Herpes Simplex Virus thymidine kinase gene placed downstream of the SIN LTR, but upstream of the vector's internal promoter, is produced in quantities high enough to render cells containing this vector partially succeptible to gancyclovir-induced cell death. Additionally, we show that SIN lentivectors containing no internal promoter express an eGFP transgene. Using RT-PCR we demonstrate that transcripts initiated at the 5' LTR in SIN lentivectors can be readily detected, and using this assay, we conclude that the SIN LTR retains approximately 15% of the transcriptional activity of the wild-type LTR (in the absence of Tat). We have also identified sequences in the SIN lentivector which are responsible for transcriptional activation at the 5' LTR. With a promoter assay using different segments of the LTR and leader region of the SIN lentivector, we have determined that the residual promoter activity does not depend on any LTR sequence, including the HIV-1 start-site initiator (Inr) element. Rather, the promoter activity is contained entirely within the leader region, and although this element is downstream of the transcription initiation site, it is capable of initiating transcription from the 5' end of R in the LTR. Mutation of leader region binding sites for the transcriptional activators DBF1 and SP1 reduces transcription from the SIN LTR by up to 80%; however, a significant decrease in vector titer results from this mutation, perhaps owing to disruption of the folding of psi, which overlaps the SP1 binding site. In conclusion, knowledge of the potential for mobilization of SIN lentivectors by HIV-1 will be important for the design of future gene therapy trials using such vectors. It is also possible that low level expression from the SIN LTR may, in some instances, be adequate for the production of small amounts of transgene in a non-cell-type specific manner, which may be relevant to studies using SIN lentivectors to introduce cell-specific expression cassettes.

342. Self-Complementary Adeno-Associated Virus 2 (AAV)-T Cell Protein Tyrosine Phosphatase Vector as a Helper-Virus To Improve Transduction Efficiency of Conventional AAV Vectors In Vitro and In Vivo

Although adeno-associated virus 2 (AAV) vectors target the liver efficiently, the transgene expression is limited to ~5% of hepatocytes. The lack of efficient transduction is due in part to the presence of a cellular protein, FKBP52, phosphorylated forms of which, inhibit the viral second-strand DNA synthesis, and consequently, transgene expression (J. Virol., 75: 8968–8976, 2001). We have documented that dephosphorylation of FKBP52 by cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV transduction of primary murine hematopoietic cells in TC-PTP-transgenic mice (J. Virol., 77: 2741–2746, 2003). Since TC-PTP cDNA size is within the packaging capacity of self-complementary AAV (scAAV) vectors, which obviate the need for viral-second-strand DNA synthesis, we reasoned that scAAV-TC-PTP vectors, if co-infected with a conventional AAV vector, might serve as a helper-virus for conventional single-stranded AAV vectors. Co-infection of HeLa cells, which are transduced poorly by conventional AAV vectors, at a 1:1 ratio with a conventional AAV-EGFP vector and scAAV vectors containing the wild-type (wt) TC-PTP gene (scAAV-wt-TC-PTP) led to a significant increase in AAV-mediated EGFP transgene expression in vitro. This increase was not observed when scAAV vectors containing a catalytically inactive mutant form of TC-PTP (scAAV-mTC-PTP) were used. We also tested the efficacy of scAAV-TC-PTP vectors in a mouse model in vivo. Approximately 1 × 1011 particles of AAV-EGFP vectors alone, or those ad-mixed with either scAAV-wtTC-PTP, or scAAV-mTC-PTP vectors were injected via the tail-vein into C57BL6 mice. Four weeks-post injection, liver tissues were harvested, sectioned and evaluated for EGFP gene expression. Consistent with previously published reports, little transgene expression occurred in hepatocytes following injection of conventional AAV-EGFP vectors, however, co-injection with scAAV-wt-TC-PTP vectors, but not with scAAV-mTC-PT-PTP vectors, led to a significant increase in EGFP expression in murine hepatocytes in vivo. Low Mr DNA samples were isolated from liver tissues, electrophoresed on 1% alkaline-agarose gels, and analyzed on Southern blots using an EGFP probe. Dimer-length AAV genomes, representing viral second-strand DNA synthesis, were detected in cells from mice co-injected with conventional AAV-EGFP+scAAV-wt-TC-PTP vectors, but not in those injected with AAV-EGFP vector alone, or co-injected with scAAV-mTC-PTP vectors. Cohorts of C57BL6 mice were also injected via the tail-vein either with PBS, or with 1 × 1011 particles of scAAV-wtTC-PTP vectors and sacrificed at the end of 4-weeks. All tissues from all PBS-injected controls, and scAAV-wtTC-PTP vector-injected animals showed no evidence of any toxicity, and no pathological lesions were observed in any organ in any of the animals. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.

534. Recombinant Adeno-Associated Virus Serotype 1 Vector-Mediated Transduction within the Murine Hippocampus

Top of pageAbstract Recent studies have shown that several of the recombinant adeno-associated virus (rAAV) serotypes have distinct biologic functions (different transduction efficiencies and cell-type tropisms) within the central nervous system. Our group reported that rAAV type 1 (rAAV1) vectors had a greater transduction efficiency than that of rAAV type 2 (rAAV2) vectors within the murine striatum. To further explore the differences between these vectors within the brain, we compared rAAV1 and rAAV2 vectors within the murine hippocampus. Methods: We engineered an rAAV type 1 hybrid vector and an rAAV type 2 vector to carry identical genomes consisting of the AAV type 2 terminal repeat elements flanking a cytomegalovirus immediate-early promoter-driven EGFP transgene. The vectors (rAAV1-GFP and rAAV2-GFP) were administrated to the brains of C3H/HeJ mice (8 weeks of age) by stereotaxic -guided injection into right hippocampus. Each mouse received 5 108 DNase resistant particles. EGFP expression was evaluated by confocal and fluorescent microscopy. Results: Mice (n = 3 for each vector type) were analyzed 5 weeks post-vector injection. In the rAAV2-GFP-injected mice, EGFP-positive cell bodies and fibers were present within the oriens layer and pyramidal cell layer surrounding the needle track (labeled by carbon black) in the CA1 area of the hippocampus. In the rAAV1-GFP-injected mice, EGFP-positive cell bodies and fibers were widely distributed in CA1, CA2, and CA3 oriens layer and pyramidal cell layer in the hippocampus ispsilateral to the injection site. Outside of the hippocampus, EGFP-positive cells were present within the septofimbrial and lateral septal nuclei (dorsal part); and EGFP-positive fibers were present within the hippocampal commissure, dorsal fornix, and dentate gyrus. Within the hemisphere contralateral to the injection site, EGFP-positive cell bodies and fibers were present within the CA1, CA2, and CA3 oriens layer and the pyramidal cell layer of the hippocampus and EGFP-positive fibers were present within the dentate gyrus. The level of EGFP expression was greater in the injected as compared to the uninjected hippocampus. EGFP (positive cell bodies and fibers) was present within the corpus callosum of both hemispheres. Serial sections through the hippocampus revealed EGFP expression extending in the rostral-caudal direction less than 0.5 mm in the rAAV2-injected mice and 2.610.34 mm in the rAAV1-injected mice. Conclusions: rAAV1 vectors have a higher transduction efficiency and more widespread distribution than rAAV2 vectors after injection into the hippocampus. Based upon the finding of EGFP-positive cell bodies within the contralateral hippocampus, rAAV1 vectors appear to undergo retrograde transportation along neuronal axons. These characteristics might prove useful in gene therapy applications targeting the hippocampus.

Targeted Correction of Single-Base-Pair Mutations with Adeno-Associated Virus Vectors under Nonselective Conditions

Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.

Long-term transgene expression in proliferating cells mediated by episomally maintained high-capacity adenovirus vectors.

High-capacity "gutless" adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo.

In a preclinical model for prostate cancer gene therapy, we have tested lentiviral vectors as a practical possibility for the transfer and long-term expression of the EGFP gene both in vitro and in vivo. The human prostate cancer cell lines DU145 and PC3 were transduced using experimental conditions which permitted analysis of the expression from a single proviral vector per cell. The transduced cells stably expressed the EGFP transgene for 4 months. After injection of the transduced cell populations into Nod-SCID mice a decrease in EGFP was only observed in a minority of cases, while the majority of tumors maintained transgene expression at in vitro levels. In vivo injection of viral vector preparations directly into pre-established subcutaneous or orthotopic tumor masses, obtained by implantation of untransduced PC3 and DU145 cells led to a high transduction efficiency. While the efficiency of direct intratumoral transduction was proportional to the dose of virus injected, the results indicated some technical limitations inherent in these approaches to prostate cancer gene therapy.

Neurofilament RNA causes neurodegeneration with accumulation of ubiquitinated aggregates in cultured motor neurons.

The mechanisms whereby mutant gene expression triggers neurodegeneration are poorly understood but have generally been attributed to translated gene products. We now demonstrate direct neuropathic effects of untranslated RNA on cultured motor neurons. We show that expression of untranslated light neurofilament (NF-L) RNA sequence in the 3'UTR of an EGFP transgene (pEGFP/NF-L RNA) or in a separate expression vector (pRc/NF-L RNA) causes dose-dependent, neuron-specific motor neuron degeneration. Neither unfused EGFP protein (pEGFP/wt) nor EGFP-tagged NF-L protein (pEGFP/NF-L protein) has similar neuropathic effects. The findings are the first demonstration of a direct RNA-mediated neurotoxic effect. Moreover, the resulting neuropathological changes show that untranslated RNA can lead to early degeneration of neuritic processes and accumulations of ubiquitinated aggregates in the perikarya and nuclei of degenerating motor neurons. The latter findings are hallmark neuropathological features of neurodegenerative diseases and their occurrence as a result of altered RNA expression raises the prospects of an RNA-mediated component in the pathogenesis of neurodegenerative states.

Alteration of Gene Expression by Chromosome Loss in the Postnatal Mouse Brain

Frequent chromosomal aneuploidy has recently been discovered in normal neurons of the developing and mature murine CNS. Toward a more detailed understanding of aneuploidy and its effects on normal CNS cells, we examined the genomes of cells in the postnatal subventricular zone (SVZ), an area that harbors a large number of neural stem and progenitor cells (NPCs), which give rise to neurons and glia. Here we show that NPCs, neurons, and glia from the SVZ are frequently aneuploid. Karyotyping revealed that approximately 33% of mitotic SVZ cells lost or gained chromosomes in vivo, whereas interphase fluorescence in situ hybridization demonstrated aneuploidy in postnatal-born cells in the olfactory bulb (OB) in vivo, along with neurons, glia, and NPCs in vitro. One possible consequence of aneuploidy is altered gene expression through loss of heterozygosity (LOH). This was examined in a model of LOH: loss of transgene expression in mice hemizygous for a ubiquitously expressed enhanced green fluorescent protein (eGFP) transgene on chromosome 15. Concurrent examination of eGFP expression, transgene abundance, and chromosome 15 copy number demonstrated that a preponderance of living SVZ and OB cells not expressing eGFP lost one copy of chromosome 15; the eGFP transgene was lost in these cells as well. Although gene expression profiling revealed changes in expression levels of several genes relative to GFP-expressing controls, cells with LOH at chromosome 15 were morphologically normal and proliferated or underwent apoptosis at rates similar to those of euploid cells in vitro. These findings support the view that NPCs and postnatal-born neurons and glia can be aneuploid in vivo and functional gene expression can be permanently altered in living neural cells by chromosomal aneuploidy.

Efficiency of chimeraplast gene targeting by direct nuclear injection using a GFP recovery assay.

Traditional RNA-DNA chimeric oligonucleotides (chimeraplasts), composed of a continuous stretch of RNA and DNA residues in a duplex conformation, have been shown to correct single-base mutations in episomal and genomic DNA both in vitro and in vivo. In the current study, we have compared the efficiency of single-base pair correction between a traditionally designed chimeraplast (covalently linked duplex) and hybrid chimeraplasts (noncovalent duplexes formed from stretches of RNA and DNA nucleotides synthesized individually and hybridized in vitro). Six hybrid chimeraplasts of identical length were constructed with various lengths of target homology and strand location of the desired nucleotide change. These constructs were evaluated for their ability to correct a point mutation in the gene encoding recombinant enhanced green fluorescent protein (eGFP) that rendered the protein nonfluorescent. A plasmid encoding this mutant eGFP gene and a chimeraplast were co-introduced directly into the nuclei of primary fibroblasts by microinjection. As shown by the recovery of eGFP fluorescence, three of the six hybrid chimeraplasts demonstrated the ability to mediate gene correction (0.4-2.4%). Covalent joining of RNA and DNA strands in chimeraplasts was not necessary for correction of DNA mutations. However, the strand placement of the desired nucleotide change and the length of nonhomologous sequences flanking target nucleotides played a crucial role in the efficiency of chimeraplast-mediated gene correction. Despite the ability of certain chimeraplast designs to correct point mutations in episomal plasmids, targeted correction of integrated copies of the mutant eGFP transgene was unsuccessful in primary fibroblasts. These results demonstrate that, although chimeraplasts are fairly effective at targeting episomal DNA in primary cells, further optimization is required to increase the efficiency for targeting integrated genes.

Expressing functional siRNAs in mammalian cells using convergent transcription

BackgroundThe use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing.ResultsWith the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells.ConclusionThe experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences. Furthermore, the cassette design reported is ideal for both routine use in controlling specific gene expression and construction of randomised RNAi expression libraries for use in unbiased forward genetic selections.

FISH Analysis of 142 EGFP Transgene Integration Sites into the Mouse Genome

Production of transgenic animals is an important technique for studying various biological processes. However, whether the integration of a particular transgene occurs randomly in the mouse genome has not been determined. Analysis by fluorescence in situ hybridization of the integration sites of the 142 EGFP (a mutant of green fluorescent protein) transgenic lines that we produced showed that the transgenes had become incorporated into every mouse chromosome. A single integration site was observed in 82.4% of the lines. The concomitant integrations of transgene into two different loci were observed in 15 cases (10.6%). In 3 cases, the transgenic founder mice showed chimerism in integration sites (2.1%). Chromosomal translocation was observed in 7 cases (4.9%). Moreover, when we statistically analyzed the transgene integration sites of these mouse lines, they were shown to distribute unevenly throughout the genome. This is the first report to analyze the transgene integration sites by producing more than 100 transgenic mouse lines.

Intracerebral transplantation and successful integration of astrocytes following genetic modification with a high-capacity adenoviral vector.

To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.

Adeno-associated virus (AAV)–mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture

Background/Purpose: Since the discovery of the cystic fibrosis transmembrane regulator (CFTR) gene, cystic fibrosis has been an attractive target for gene therapy. Postnatal gene transfer in the respiratory epithelium has been difficult and particularly inefficient in the submucosal gland cells, the target cells for CFTR gene transfer. The authors hypothesized that during development, there is a favorable environment for fetal gene therapy with fewer physical barriers to efficient gene transfer and more accessible epithelial and submucosal gland precursor cells. The authors tested this hypothesis in a novel human fetal tracheal organ culture system using a serotype 2 recombinant AAV that contains an enhanced green fluorescent protein (eGFP) reporter gene (AAV-CMV-eGFP). Methods: Human fetal tracheas at between 16 and 20 weeks' gestation age were used in this study. The distal end of each trachea was ligated and secured in an upright position with the open proximal end facing up. Only the ante-lumenal surface was exposed to culture media. 5 × 109 particles of AAV-CMV-eGFP were administered intratracheally through the open end. Fetal tracheas were maintained in tracheal organ culture media and harvested at either 2 weeks (n = 3) or 4 weeks (n = 7) after injection. The fetal tracheas were processed for routine H&E, standard electron microscopy (EM), and fluorescence microscopy for analysis of eGFP transgene expression. Results: Histology confirmed the preservation of structural integrity out to 4 weeks of fetal tracheal organ culture. EM showed intact tight junctions of the apical respiratory epithelium. At 2 weeks after AAV-CMV-eGFP injection, there was minimal transgene expression. However, at 4 weeks there was extensive transgene expression in not only the respiratory surface epithelium but also the submucosal gland cells of the human fetal tracheal organ culture. Transgene expression was seen in nearly all cells in the submucosal glands. Conclusions: AAV-mediated gene transfer in human fetal tracheal organ culture was highly efficient with excellent transgene expression at 4 weeks in both respiratory surface epithelium and submucosal gland cells. This highly efficient gene transfer may result from fewer physical barriers and more accessible target precursor cells. These results are consistent with more efficient gene transfer in fetal tracheobronchial epithelium and show the potential for fetal gene therapy using AAV for the treatment of congenital airway disease such as cystic fibrosis. J Pediatr Surg 37:1051-1057. Copyright 2002, Elsevier Science (USA). All rights reserved.

DEVELOPMENT OF A TRANSGENIC GREEN FLUORESCENT PROTEIN LINEAGE MARKER FOR STEROIDOGENIC FACTOR 1

Knockout (KO) mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired function of pituitary gonadotropes, and abnormalities of the ventromedial hypothalamic nucleus (VMH). To develop a lineage marker for cells that express SF-1, we used bacterial artificial chromosome (BAC) transgenesis. A BAC fragment containing 50 kb of the mouse Nr5a1 gene was placed upstream of the coding sequence for enhanced green fluorescent protein (eGFP) and used to generate SF-1/eGFP transgenic mice. These sequences directed eGFP expression to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, VMH neurons, and reticuloendothelial cells of the spleen. Despite the essential role of SF-1 in gonadotropes, eGFP was not expressed in the anterior pituitary. These studies show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgene provides a valuable tool to expand our understanding of the actions of SF-1 in endocrine development and function.

Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos.

Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal-fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture of in vitro fertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription-PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.

A CD2-green fluorescence protein-transgenic mouse reveals very late antigen-4-dependent CD8+ lymphocyte rolling in inflamed venules.

Intravital microscopy allows detailed analysis of leukocyte trafficking in vivo, but fails to identify the nature of leukocytes investigated. Here, we describe the development of a CD2-enhanced green fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte trafficking during inflammation in vivo. A CD2-EGFP plasmid construct including the CD2 promoter, the EGFP transgene, and the CD2 locus control region was injected into B6CBA/F1 pronuclei. EGFP+ offspring were backcrossed into C57BL/6 mice for six generations. Flow cytometry demonstrated that all peripheral blood EGFP+ cells were positive for CD2 and negative for the granulocyte Ag Ly 6-G (GR-1). EGFP(high) cells stained positive for CD2, CD3, CD8, TCR beta-chain, and NK1.1 but did not express the B cell and monocyte markers CD45RA, CD19, and CD11b. In vitro stimulation assays revealed no difference in lymphocyte proliferation and IL-2 secretion between EGFP+ and EGFP- mice. Intravital microscopy of untreated or TNF-alpha-treated cremaster muscle venules showed EGFP+ cells in vivo, but these cells did not roll or adhere to the vessel wall. In cremaster muscle venules treated with both TNF-alpha and IFN-gamma, EGFP(high) cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 microm/s) than that of neutrophils (10 microm/s). Blocking alpha4 integrin with a mAb increased rolling velocity to 24 microm/s. These findings show that CD8+ T cells roll in TNF-alpha/IFN-gamma-pretreated vessels in vivo via an alpha4 integrin-dependent pathway.

Enhanced Transgene Expression in Cord Blood CD34+-Derived Hematopoietic Cells, Including Developing T Cells and NOD/SCID Mouse Repopulating Cells, Following Transduction with Modified TRIP Lentiviral Vectors

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34+ cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3′ LTR in the TRIP-CMV vector resulted in a safer vector (TRIPΔU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1α (EF1α). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34+-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPΔU3-EF1α, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34+-derived T, B, NK, and myeloid hematopoietic cells.

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

Multipotent Stem Cells from the Brain and Retina of Green Mice

THE RECENT ISOLATION AND AMPLIFICATION of multipotent stem cells in the laboratory (1,2) has enlivened the fields of mammalian development and transplantation. Yet the very plasticity that makes these cells so interesting biologically makes them difficult to track as they integrate into host tissues. Here we describe the isolation of stem cells from the brain and retina of mice expressing the enhanced green fluorescent protein (eGFP) transgene, and the transplantation of these cells to the brain and retina of nontransgenic recipients. The most successful method for labeling stem cells has been transfection with a heritable reporter gene such as beta-galactosidase (b-GAL) (3) or GFP (4). These techniques are performed following isolation and prior to grafting. They are slow, labor-intensive, and require transfection of each cell type isolated, as well as repeated clonal selection for high level expression. Loss of transgene expression following differentiation or transplantation is an additional problem with these techniques. Furthermore, each transfection alters the genome unpredictably, risking unintentional changes in stem cell phenotype. While direct isolation of stem cells from b-Gal1 mice has been described, this label has the disadvantage of requiring an exogenous substrate for visualization. By harvesting from donors transgenic for GFP, it should be possible to obtain stem cell lines exhibiting uniformly high levels of constitutive reporter gene expression. Transgenic animals expressing GFP throughout their body have been generated in a number of species, including mice (5–7). We harvested tissue from newborn GFP transgenic mice and isolated stem cells from the brain (BSCs) and neuroretina (RSCs) using established methods (20 ng/mL EGF and bFGF). In both cases, GFP1 neurospheres appeared within the first 3 days and have now been maintained in culture for more than 1 year. To assess the capacity for self-renewal, spheres were broken up and plated as single cells. Individual cells formed new spheres over a period of 2–5 days, with number and size proportional to time in culture. Spheres labeled positively for nestin, an intermediate filament expressed by neuronal and glial precursors, and for Ki-67, which recognizes actively mitotic cells (Fig. 1a–1c). Spheres cultured in the presence of 10% fetal bovine serum generated cells that maintained their green fluorescence and co-labeled with neural markers. BSCs differentiated into cells labeled with markers of each neural lineage; that is, neurons (MAP-2), astrocytes (GFAP) and oligodendrocytes (GalC) (Fig. 1d–1f). RSCs did not differentiate into oligodendrocytes, a cell type not normally generated in the retina, but did differentiate into cells expressing the photoreceptor-specific marker rhodopsin. Green mouse neurospheres were injected into sites within the brain and eye of mature mice. Transplanted BSCs survived, maintained GFP expression (as evidenced by co-labeling after pulsing with BrdU in vitro), migrated widely and showed morphological evidence of integration with the host central nervous system (Fig. 1g). RSCs grafted to the subretinal space of the mechanically injured mouse retina showed morpho-