Biomedical subjects

B Zheng

Publications and source records attributed to B Zheng.

At least 109 records · Page 6Linked to original sources

PubMed1998

Isolation of yeast mutants defective for localization of vacuolar vital dyes.

An application of flow cytometric sorting is used for isolation of Saccharomyces cerevisiae mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrie nyl )pyridinium dibromide (FM4-64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15 degreesC. Cells stained at 15 degreesC for both FM4-64 and carboxydichlorofluorescein diacetate (a vacuolar luminal vital stain), had a pronounced shift in red/green fluorescence from cells stained at 30 degrees or 38 degreesC. Flow cytometric selection based on this characteristic shift allowed the isolation of 16 mutants. These comprised 12 complementation groups, which we have designated SVL for styryl dye vacuolar localization. These groups were put into three classes. Class I mutants contain very large vacuoles; class II mutants have very fragmented vacuoles; and class III mutants show the strongest svl phenotype with punctate/diffuse FM4-64 staining. Limited genetic overlap was observed with previously isolated mutants, namely svl2/vps41, svl6/vps16, and svl7/fab1. The remaining svl mutants appear to represent novel genes, two of which showed temperature-sensitive vacuole staining morphology. Another mutant, svl8, displayed defects in uptake and sorting of phosphatidylcholine and phosphatidylethanolamine. Our flow cytometric strategy may be useful for isolation of other mutants where mislocalization of fluorescent compounds can be detected.

Biological Transport, Active↗

PubMed1998

Blocking ligand occupancy of the alphaVbeta3 integrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells.

Blocking alphaVbeta3 integrin occupancy results in attenuation of the cellular migration response to insulin-like growth factor I (IGF-I). To determine whether integrin antagonists alter other IGF-I-stimulated biologic actions, quiescent smooth muscle cells (SMCs) were exposed to echistatin and their ability to respond to IGF-I was determined. Echistatin (10(-7) M) inhibited IGF-I-stimulated DNA synthesis by 80%, and the protein synthesis response also was inhibited. Therefore blocking occupancy of alphaVbeta3 inhibited multiple target cell actions of IGF-I. To determine whether blocking alphaVbeta3 occupancy could alter IGF-I receptor-mediated signal transduction, the ability of IGF-I to stimulate phosphorylation of insulin receptor substrate-1 (IRS-1) was analyzed. A 10-min exposure to 100 ng/ml of IGF-I resulted in a substantial increase in phosphorylated IRS-1, and echistatin (10(-7) M) blocked the IGF-I-induced IRS-1 phosphorylation response. Echistatin also attenuated downstream signaling because the capacity of the p85 subunit of phosphatidylinositol-3 kinase (PI-3 kinase) to bind to IRS-1 was blocked. In contrast, exposure of SMCs to vitronectin (1.0 micrograms/cm2) or thrombospondin (0.25 micrograms/cm2), two known ligands for alphaVbeta3, resulted in enhancement of the IGF-I-stimulated IRS-1 response. To determine whether these effects were caused by alterations in receptor kinase activity, the IGF-I receptor was immunoprecipitated and then analyzed for phosphotyrosine. Echistatin (10(-7) M) significantly reduced IGF-I-stimulated tyrosine phosphorylation of the IGF-I receptor beta subunit. We conclude that occupancy of the alphaVbeta3 integrin is necessary for IGF-I to fully activate the kinase activity of the IGF-I receptor and phosphorylate IRS-1. Activation of the alphaVbeta3 receptor results in an interaction with the IGF-I signal transduction pathway, which modulates SMCs responsiveness to IGF-I.

Animals↗

PubMed1998

The effect of extracellular matrix proteins on porcine smooth muscle cell insulin-like growth factor (IGF) binding protein-5 synthesis and responsiveness to IGF-I.

The aim of this study was to determine if cultured porcine vascular smooth muscle cells (pSMCs) that had been maintained on different extracellular matrix proteins had an alteration in their expression of insulin-like growth factor binding protein-5 (IGFBP-5) and their responsiveness to insulin-like growth factor-I (IGF-I). When pSMCs were plated on fibronectin, they synthesized 6.0 +/- 1.2-fold more IGFBP-5 than did cells maintained on laminin and type IV collagen. IGF-I increased IGFBP-5 gene expression 3-fold in the cells plated on fibronectin. The addition of an RGD peptide and echistatin to pSMC cultures that had been plated on fibronectin inhibited IGFBP-5 mRNA expression. The addition of an antibody against alpha2beta1 integrin partially reversed the inhibitory effects of laminin and type IV collagen on IGFBP-5 expression. Cells maintained on fibronectin had a 5.0 +/- 1.1-fold greater DNA synthesis response to IGF-I compared with those maintained on laminin/type IV collagen, and echistatin significantly inhibited the DNA synthesis response of the fibronectin-maintained cells to IGF-I. The anti-alpha2beta1 antibody partially reversed the inhibitory effect of laminin and type IV collagen on IGF-I-stimulated DNA synthesis. The addition of IGFBP-5 to cultures plated on laminin and type IV collagen significantly increased their response to IGF-I. Atherosclerotic plaques from pig aorta contained abundant fibronectin and had increased IGFBP-5 mRNA (4.5 +/- 1.5-fold) compared with tissue from the normal vessel wall that had a low fibronectin content. These results indicate that fibronectin, laminin, and type IV collagen have major effects on IGFBP-5 expression and on IGF-I-stimulated pSMC responses and that these effects are mediated by their respective integrins.

Animals↗

PubMed1998

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

Animals↗

PubMed1998

Incorporation of a set enumeration trees-based classifier into a hybrid computer-assisted diagnosis scheme for mass detection.

RATIONALE AND OBJECTIVES: The authors evaluated whether a hybrid classifier of two independent computer-aided diagnosis (CAD) schemes, the set enumeration (SE) trees approach and an artificial neural network (ANN), could improve the detection of masses on digitized mammograms. The potential benefits resulting from the interpretability of the SE trees model was also explored. MATERIALS AND METHODS: Two hundred thirty verified mass regions and 230 negative but suspicious regions were randomly selected from 618 digitized mammograms. Each region was represented by a 24-parameter feature vector. These features were used as input data for the SE trees and ANN-based schemes. After the positive and negative regions were randomly segmented into five exclusive partitions, a fivefold cross-validation method was applied to evaluate and compare the performance of the SE trees, ANN, and hybrid system in the identification of masses. RESULTS: The performance of the SE trees approach was comparable to that of the ANN. The average area under the receiver operating characteristic (ROC) curves for all five partitions was 0.88 (standard deviation, 0.04). Owing to the relatively low correlation between the region-based results of the SE trees and ANN methods, the hybrid classifier yielded a significantly improved performance, with an area under the ROC curve of 0.94 (standard deviation, 0.02; P < .05). CONCLUSION: The hybrid CAD scheme significantly improved performance. The amenability of the SE trees models to interpretation may aid in the assessment of the importance of specific features.

Artificial Intelligence↗

PubMed1998

Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair.

Germline mutations in the human MSH2, MLH1, PMS2 and PMS1 DNA mismatch repair (MMR) gene homologues appear to be responsible for most cases of hereditary non-polyposis colorectal cancer (HNPCC; refs 1-5). An important role for DNA replication errors in colorectal tumorigenesis has been suggested by the finding of frequent alterations in the length of specific mononucleotide tracts within genes controlling cell growth, including TGF-beta receptor type II (ref. 6), BAX (ref. 7) and APC (ref. 8). A broader role for MMR deficiency in human tumorigenesis is implicated by microsatellite instability in a fraction of sporadic tumours, including gastric, endometrial and colorectal malignancies. To better define the role of individual MMR genes in cancer susceptibility and MMR functions, we have generated mice deficient for the murine homologues of the human genes MLH1, PMS1 and PMS2. Surprisingly, we find that these mice show different tumour susceptibilities, most notably, to intestinal adenomas and adenocarcinomas, and different mutational spectra. Our results suggest that a general increase in replication errors may not be sufficient for intestinal tumour formation and that these genes share overlapping, but not identical functions.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Identification of clustered microcalcifications on digitized mammograms using morphology and topography-based computer-aided detection schemes. A preliminary experiment.

RATIONALE AND OBJECTIVES: A mathematical morphology-based computer-aided detection (CAD) scheme for the identification of clustered microcalcifications was developed and tested. The potential for improving either sensitivity or specificity by combining the results with those previously reported was investigated. METHODS: The CAD scheme presented here is based on mathematical morphology and a series of simple rule-based criteria for the identification of clustered microcalcifications. A database of 105 digitized mammograms was used for training and rule setting of the scheme. A test set of 191 digitized mammograms was used to evaluate its performance. The same test set had been used to evaluate a multilayer, topography-based scheme. The results obtained by the two schemes were then combined using logical OR and AND operations. RESULTS: The morphology-based and topography-based CAD schemes performed at sensitivities of 82.9% and 89.5%, with false-positive detection rates of 1.3 and 0.4 per image, respectively. A logical OR operation resulted in 95.4% sensitivity. An AND operation achieved 76.2% sensitivity, with no false identifications on 93% of images. CONCLUSIONS: By combining the results of the morphology-based and the topography-based schemes, either sensitivity or specificity can be improved.

Algorithms↗

PubMed1998

Immunoglobulin gene hypermutation in germinal centers is independent of the RAG-1 V(D)J recombinase.

Antigen-driven somatic hypermutation in immunoglobulin genes coupled with stringent selection leads to affinity maturation in the B-lymphocyte populations present in germinal centers. To date, no gene(s) has been identified that drives the hypermutation process. The site-specific recombination of antigen-receptor gene segments in T and B lymphocytes is dependent on the expression of two recombination activating genes, RAG-1 and RAG-2. The RAG-1 and RAG-2 proteins are essential for the cleavage of DNA at highly conserved recombination signals to make double-strand breaks and their expression is sufficient to confer V(D)J recombination activity to non-lymphoid cells. Until very recently, expression of the V(D)J recombinase in adults was believed to be restricted to sites of primary lymphogenesis. However, several laboratories have now demonstrated expression of RAG-1 and RAG-2 and active V-to-(D)J recombination in germinal center B cells. This observation of active recombinase in germinal centers raises the issue of RAG-mediated nuclease activity as a component of V(D)J hypermutation. Here, we show that a transgenic kappa-light chain gene in a RAG-1-/- genetic background can acquire high frequencies of mutations. Thus, the RAG-1 protein is not essential for the machinery of immunoglobulin hypermutation. The genetic approaches to identifying the genes necessary for somatic hypermutation will require further studies on DNA-repair and immunodeficient models.

Animals↗

PubMed1998

Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by fibroblasts and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys120-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.

Animals↗

PubMed1998

[Studies on mutagenicity of humic acid samples collected from arsenosis-affected areas in China].

OBJECTIVE: To explore whether there is mutagenicity of humic acid samples collected from arsenosis affected areas in China. METHODS: Ames' test was used for natural humic acid samples extracted from the coal containing high arsenic in Guizhou Province, and the well water in the areas with blackfoot disease in Taiwan and with arsenosis in Inner Mongolia of China. RESULTS: Coefficients of revertant mutation were 1.02-0.82 net revertant per microgram (rev/microgram) of humic acid extracted from the coal containing high arsenic in Guizhou without rat liver homogenate (-S9), and 1.53-1.12 rev/microgram of that with rat liver homogenate (+S9), with an obvious dose-response relationship. CONCLUSION: Humic acid extracted from the coal containing high arsenic produced in Guizhou could cause stronger direct mutation in Salmonella typhimurium TA98 (frameshift mutation) and that extracted from the well water in the areas with blackfoot disease in Taiwan could cause weaker direct mutation in Salmonella typhimurium (-S9), but that extracted from the well water with arsenosis in Inner Mongolia could not cause mutation. Mutagenicity of commercial humic acid, purchased from Aldrich Co. in the United States, arsenic and iron was also studied, and no mutagenicity was found for them independently, but arsenic combined with humic acid, at certain concentrations, could cause weak mutation in TA98 (-S9), with or without iron. No mutagenicity in TA100 (+/- S9, base pair substitution mutation) was found in all the samples mentioned above.

Animals↗

PubMed1998

Thirteen years of manipulating the mouse genome: a personal history.

In 1974, Dr. Ralph Brinster published a paper describing the consequences of injecting embryonal carcinoma cells, the predecessors of embryonic stem cells, into mouse blastocysts. Despite their early promise, embryonal carcinoma cells would not efficiently populate the germ line of mice. A decade later Elizabeth Robertson and I described the efficient generation of germline chimaeras from cultured embryonic stem cells and shortly afterwards the genetic manipulation of the mouse germline using ES cells. Our demonstration of the potency of Embryonic Stem cells gave birth to a new era in manipulative mouse genetics, one in which endogenous genes can now be mutated at will using gene targeting of retroviral mutagenesis. This review focuses on the development and testing of concepts and techniques during the thirteen years after we knew germline modification of endogenous genes in the mouse would be possible. This period is one in which more and more sophisticated tools for manipulating the mouse germline were developed and implemented. In this review I have taken the rare opportunity to reveal some of my thought processes, frustrations, successes and failures as we moved through this exciting period of rapid technological change. As I look forward to the next thirteen years, I feel that this will be an equally exciting period for manipulative genetics as we struggle to formulate concepts and design experiments that enable us to understand gene function in an era when the sequence of all genes will be known.

Animals↗

PubMed1997

V(D)J recombinase activity in a subset of germinal center B lymphocytes.

Reexpression of the V(D)J recombinase-activating genes RAG1 and RAG2 in germinal center B cells creates the potential for immunoglobulin gene rearrangement and the generation of new antigen receptor specificities. Intermediate products of V(D)J recombination are abundant in a subset of germinal center B cells, demonstrating that the kappa immunoglobulin light-chain locus becomes a substrate for renewed V(D)J recombinase activity. This recombinationally active cell compartment contains many heavy-chain VDJ rearrangements that encode low-affinity or nonfunctional antibody. In germinal centers, secondary V(D)J recombination may be induced by diminished binding to antigen ligands, thereby limiting abrupt changes in receptor specificity to B cells that are usually eliminated from the germinal center reaction. This restriction preserves efficient antigen-driven selection in germinal centers while allowing for saltations in the somatic evolution of B cells.

Animals↗

PubMed1997

Characterization of a novel mammalian RGS protein that binds to Galpha proteins and inhibits pheromone signaling in yeast.

Genetic studies of molecules that negatively regulate G-coupled receptor functions have led to the identification of a large gene family with an evolutionarily conserved domain, termed the RGS domain. It is now understood that RGS proteins serve as GTPase-activating proteins for subfamilies of the heterotrimeric G-proteins. We have isolated from mouse pituitary a full-length cDNA clone encoding a novel member of the RGS protein family, termed RGS16, as well as the full-length cDNA of mRGS5 and mRGS2. Tissue distribution analysis shows that the novel RGS16 is predominantly expressed in liver and pituitary, and that RGS5 is preferentially expressed in heart and skeletal muscle. In contrast, RGS2 is widely expressed. Genetic analysis using the pheromone response halo assay and FUS1 gene induction assay show that overexpression of the RGS16 gene dramatically inhibits yeast response to alpha-factor, whereas neither RGS2 nor RGS5 has any discernible effect on pheromone sensitivity, pointing to a possible functional diversity among RGS proteins. In vitro binding assays reveal that RGS5 and RGS16 bind to Galphai and Galphao subunits of heterotrimeric G-proteins, but not to Galphas. Based on mutational analysis of the conserved residues in the RGS domain, we suggest that the G-protein binding and GTPase-activating protein activity may involve distinct functional structures of the RGS proteins, indicating that RGS proteins may exert a dual function in the attenuation of signaling via G-coupled receptors.

Amino Acid Sequence↗

PubMed1997

Using targeted large deletions and high-efficiency N-ethyl-N-nitrosourea mutagenesis for functional analyses of the mammalian genome.

The Human Genome Project has generated nucleotide sequences from an estimated 80,000 to 100,000 genes, only a small fraction of which have a known role. Nucleotide sequence information alone is insufficient to predict gene function. One of the most powerful ways of revealing gene function, as demonstrated in bacteria, worms, yeast, and flies, is to generate mutations and characterize them at both the phenotypic and the molecular levels. Given the physiological and anatomical parallels between mouse and human, genotype-phenotype relationships established in mice can be extrapolated to human syndromes. A new method is described for functional genetic analyses in the mouse that uses loxP/Cre engineering to generate coat color-tagged large deletions. The haploid regions can then be dissected by mutagenesis with N-ethyl-N-nitrosourea in phenotype-driven screens to obtain functional information on genes in any desired region of the mouse genome.

Animals↗

PubMed1997

Distinctive characteristics of germinal center B cells.

A cardinal property of the immune system is its ability to respond to an antigen that was encountered years before with an accelerated and enhanced secondary response. The property of anamnestic reactions depends upon the formation of long-lived compartments of specialized T and B lymphocytes called memory cells. While the origin of the memory T-cell compartment is not known, germinal centers are the specialized sites for memory B-cell generation and the immunoglobulin V-region hypermutation necessary for the affinity maturation of serum antibody. Interestingly, the peripheral differentiation pathway that leads to this most mature B-cell state begins with the recapitulation of many characters of immature B lymphocytes in bone marrow. This review describes the distinctive cellular basis of germinal center reaction and the characteristics of B cells in germinal centers that later enter the memory pool.

Animals↗

PubMed1997

Computer-aided detection of clustered microcalcifications on digitized mammograms: a robustness experiment.

RATIONALE AND OBJECTIVES: The authors assessed the performance of an existing computer-aided diagnosis (CAD) scheme for the detection of clustered microcalcifications in a large image database. METHODS: A previously developed, rule-based system was used to assess detectability of microcalcification clusters in a set of 386 digitized mammograms with 239 verified clusters visible on 191 images. The test was performed without any reoptimization of the scheme. None of the 386 images had been used in any previous scheme development or testing procedures. RESULTS: The CAD scheme achieved 89.5% sensitivity at an average false-positive detection rate of 0.39 per image. In 75% of all images, no false-positive findings occurred. Twenty-three of 25 false-negative findings (misses) occurred during the last two stages in the detection process. CONCLUSION: This scheme produced reasonable results in a large data set of images with a large variety of cluster characteristics.

Calcinosis↗

PubMed1997

Adequacy testing of training set sample sizes in the development of a computer-assisted diagnosis scheme.

RATIONALE AND OBJECTIVES: The authors assessed the performance changes of a computer-assisted diagnosis (CAD) scheme as a function of the number of regions used for training (rule-setting). MATERIALS AND METHODS: One hundred twenty regions depicting actual masses and 400 suspicious but actually negative regions were selected as a testing data set from a database of 2,146 regions identified as suspicious on 618 mammograms. An artificial neural network using 24 and 16 region-based features as input neurons was applied to classify the regions as positive or negative for the presence of a mass. CAD scheme performance was evaluated on the testing data set as the number of regions used for training increased from 60 to 496. RESULTS: As the number of regions in the training sets increased, the results decreased and plateaued beyond a sample size of approximately 200 regions. Performance with the testing data set continued to improve as the training data set increased in size. CONCLUSION: A trend in a system's performance as a function of training set size can be used to assess adequacy of the training data set in the development of a CAD scheme.

Breast Neoplasms↗

PubMed1997

Increment of hFIX expression with endogenous intron 1 in vitro.

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence. hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i'IX, retroviral vector GINaCi'IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKGoi'IX, 151 ng/10(6) cells/24h; PA317/G1NaCi'IX, 308 ng/10(6) cells/24 h; C2C12/G1 NaCi'IX, 188 ng/10(5) cells/24 h; RSF/G1NaCi'IX, 1929 ng/10(5) cells/24 h; HSF/G1NaCi'IX, 1646 ng/10(6) cells/24 h. These results indicated that hFIX minigene with intron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production, a retroviral vector G1NaCi'IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/10(6) cells/24 h with 79% of bioactivity. PCR detection of HT/G1NaCi'IXR cells infected with PA317/G1NaCi'IXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intron1-carrying hFIX expression cassette can be used in directed gene transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.

Animals↗