Development of a retrovirus-based complementary DNA expression system for the cloning of tumor antigens

A new retroviral system has been developed for the generation of a cDNA library and the functional cloning of tumor antigens. These retroviral vectors contain a cytomegalovirus promoter in the 5' long terminal repeat, an extended packaging signal for rapid production of high-titer retroviral particles, and many convenient cloning sites for cDNA library construction. The vesicular stomatitis virus G protein has been used to generate pseudotype retroviral particles to enable efficient viral infection. Using this system, viral titers in the range of 10(6) colony-forming units/ml could be generated routinely, and a high transduction efficiency in human primary cells, including fibroblasts, was achieved. In addition, a new procedure has been devised for screening a retrovirus-based cDNA library without a functional selection. The utility of this system was demonstrated by constructing a retrovirus-based cDNA library and re-isolating the NY-ESO-1 tumor antigen from a cDNA library using an antigen-specific CTL. This approach can facilitate the identification of novel tumor antigens recognized by T cells without knowledge of MHC class I restriction elements and is generally applicable for the isolation of any gene as long as a biological assay is available.     

Gene expression of lipid binding protein transferred the ability of specific attachment of hemopoietic cells to non-supportive stromal cell line, MS-K

In order to identify the cDNAs responsible for hemopoietic supportive activity, expression of mRNAs in hemopoietic supportive bone marrow stromal cells (MS-5) and non-supportive stromal cells (MS-K) were compared by the cDNA subtraction method. A subtracted MS-5-cDNA library, which contains cDNA clones corresponding to mRNAs present in MS-5 cells but not in MS-K cells, was constructed. Screening of subtracted MS-5-cDNA library resulted in the isolation of some clones. Two of them, lipid binding protein (LBP) and haptoglobin (Hp), were expressed specifically in MS-5 cells but not in MS-K cells. The genes of LBP and Hp were subcloned into mammalian expression vector and transfected into hemopoietic non-supportive stromal cells line, MS-K. Then LBP-expressing stable transformants (MS-K-LBP) and Hp-expressing transformants (MS-K-Hp) were cloned. A rosette formation assay was carried out to investigate whether or not the LBP and Hp cause MS-K cells to adhere to hemopoietic cells. MS-K-LBP formed rosettes with hemopoietic cells as MS-5 cells, although the MS-K-Hp and normal MS-K cells did not form rosettes. These data indicate that LBP expressed in hemopoietic supportive stromal cells is partly responsible for the adhesion of hemopoietic stem cells to stromal cells.     

Use of polymerase chain reaction in analysing recombinant cDNA clones encoding storage proteins of chickpea Cicer arietinum L

A simple and reliable method was undertaken for the use of polymerase chain reaction in analyzing cDNA clones. Amplification was done of the inserts from positive legumin clones isolated from a cDNA library constructed from developing chickpea cotyledons in the expression vector, gt11. Amplification was made simple by using oligonucleotide primers which allowed convenient sizing, subcloning and sequencing of inserts by di-deoxy chain termination method. This simple method may provide opportunity to isolate large number of agronomically important genes from gene libraries.     

Generation of cDNA expression libraries enriched for in-frame sequences

Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.     

Isolation and refined regional mapping of expressed sequences from human chromosome 21

To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3.     

Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias.     

Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection

We used genetic strategies which have been proven valuable to decipher signaling pathways in comparatively simple organisms such as Drosophila and Caenorhabditis elegans, to dissect signaling network activated by tyrosine kinases in mammals. The strategy was developed further towards a generally applicable expression cloning system to identify signal transducers in tyrosine kinase pathways. This system is based on the ability of downstream acting genes to rescue the transformation phenotype of partial loss-of-function mutants of BCR-ABL which still retain tyrosine kinase activity. Using this strategy we have previously shown that overexpression of c-Myc and Cyclin D1 can rescue a signaling defective SH2 mutant of BCR-ABL for transformation. In an unbiased approach to identify new compensating genes, a cDNA library was introduced by retroviral infection into fibroblasts which express the BCR-ABL SH2 mutant. CDNA clones, capable of rescuing the SH2 mutant for transformation should result in colony formation in soft agar. A PCR approach was used to recover these compensating genes from the genomic DNA of the transformed fibroblasts. Sequencing analysis of the initial cDNAs identified three known genes, the adapter molecule Shc, the kinases SPRK and p38 MAPK. These genes have been found to interact functionally with BCR-ABL for fibroblast and hematopoietic cell transformation. Currently, we are constructing and screening new libraries to identify novel genes which complement the BCR-ABL SH2 mutant. Our results demonstrate that this cloning approach is an effective means of identifying and characterizing signaling molecules that function in specific signaling pathways. This in turn may identify specific targets for mechanism-based therapeutic intervention to block altered signaling.     

Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells

We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4+ T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4+ peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.     

Cell-to-cell transfer of glycosylphosphatidylinositol-anchored membrane proteins during sperm maturation

The majority of gene therapy protocols have used or plan to use retroviral vectors based upon murine leukaemia virus. These vectors are able to infect many different cell types, and the retroviral promoter, which is often used to control the expression of a therapeutic gene, is active in a wide range of different cell types. Safe and efficient gene transfer systems, whether based upon retroviruses or other agents, should deliver beneficial genes only to cells that require their therapeutic action, and these genes ideally should be expressed exclusively in such cells. In this paper, strategies for redirecting the infection spectrum of retroviral vectors in order to obtain cell-targeted gene delivery are discussed. These strategies include the engineering of the retroviral envelope protein, which, together with the availability of its cognate receptor, determines infectivity, and the use of proteins from other enveloped viruses of both retroviral and nonretroviral origin in the cell lines used to produce retroviral vector virus particles. Expression targeting can be achieved by limiting the expression of therapeutic genes to the cell type(s) of interest using promoters from genes that are normally active in these cells. This approach to targeting is illustrated using promoters from genes expressed in either the liver, the pancreas or the mammary gland as a means to limit gene expression specifically to the cell types that make up these organs. The successful utilization of new generations of targeted retroviral vectors in the clinic may well pave the way for superior gene delivery systems of the future that seek out their target cell, delivering a therapeutic gene to and expressing it only in such cells.     

Changing patterns of gene expression identify multiple steps during regression of rat prostate in vivo

The rat ventral prostate is an androgen-dependent organ that undergoes dramatic cell death upon removal of testosterone by surgical castration. Several well characterized criteria, such as nuclear condensation, organelle blebbing, and DNA fragmentation, have been used to demonstrate that most of this cell loss is due to programmed cell death, or apoptosis, of the secretory epithelial cells. In addition to changes in morphology, it is well known that cells undergoing apoptosis show alterations in gene expression, and it is widely assumed that many of these genes are directly involved in the mechanism of programmed cell death. Using poly A+ RNA derived from normal rat prostate as well as from the regressing prostates of castrated rats, we have used a PCR-based subtractive hybridization approach to generate complementary DNA (cDNA) libraries greatly enriched in cDNAs strongly regulated during rat prostate regression. Several hundred of the genes represented in these libraries appear to be strongly regulated during prostate regression and most of these are prostate specific. Sequence analysis indicates that up to 30% of these clones are similar or identical to genes of known function, approximately 20% are similar to expressed sequence tags (ESTs), and as many as 50% of these clones have not been characterized previously. Analysis of selected clones using in situ hybridization indicates that they are expressed specifically in prostate epithelial cells, and that certain of these clones are regulated temporally in a pattern consistent with apoptosis. The patterns of gene expression include: 1) genes whose expression decreases uniformly after removal of androgen, indicative of androgen sensitive genes; 2) genes whose expression increases in apoptotic prostate cells and in other tissues, suggesting a class of genes generally involved in apoptosis; 3) and genes whose expression increases in individual regressing prostate epithelial cells, suggesting a class of prostate specific genes associated with apoptosis.     

The spread of a replication-competent MuLV retroviral vector can be efficiently blocked by deletion variants

A retroviral vector in which the gag and pol genes have been replaced by the NLS-lacZ reporter gene was derived from a cloned AKV-like virus. A complementing cell line expressing the gag and pol retroviral genes was constructed. The retroviral vector was demonstrated to replicate in the complementing cells. Since transfection is known to generate deletion variants of the introduced plasmid, we have examined whether it can give rise to viral forms with a replicating advantage over the initial vector. After transfection in complementing cells the spread of the vector was followed by X-gal staining. The fraction of stained cells increased for the first 10 days following transfection and was then stabilized to about 20% stained cells, thus defining two cell types; one with LacZ+ phenotype and one with LacZ- phenotype. Molecular analysis showed that the latter contains a deleted form of the virus preventing cell infection by the vector presumably through a mechanism of interference involving the viral env gene. Thus, interference results in the efficient block of vector expansion.     

Measurement of aggression in psychiatric patients

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.     

Development of a yeast trihybrid screen using stable yeast strains and regulated protein expression

We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling.