Showing papers on "Transgene published in 1986"

Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector

Abstract: Embryonic stem cells isolated directly from mouse embryos1 can be cultured for long periods in vitro and subsequently repopulate the germ line in chimaeric mice2,3. During the culture period these embryonic cells are accessible for experimental genetic manipulation4–6. Here we report the use of retroviral vectors to introduce exogenous DNA sequences into a stem-cell line and show that these modified cells contribute extensively to the somatic and germ-cell lineages in chimaeric mice. Compared with current methods for manipulation of the mouse genome, this approach has the advantage that powerful somatic-cell genetic techniques can be used to modify and to select cells with germ-line potential, allowing the derivation of transgenic strains with pre-determined genetic changes. We have by this means inserted many proviral vector sequences that provide new chromosomal molecular markers for linkage studies in the mouse and that also may cause insertional mutations.

The hypogonadal mouse: reproductive functions restored by gene therapy.

Abstract: The hypogonadal (hpg) mouse lacks a complete gonadotropin-releasing hormone (GnRH) gene and consequently cannot reproduce. Introduction of an intact GnRH gene into the genome of these mutant mice resulted in complete reversal of the hypogonadal phenotype. Transgenic hpg/hpg homozygotes of both sexes were capable of mating and producing offspring. Pituitary and serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin were restored to those of normal animals. Immunocytochemistry and in situ hybridization showed that GnRH expression was restored in the appropriate hypothalamic neurons of the transgenic hpg animals, an indication of neural-specific expression of the introduced gene.

Functional analysis of regulatory elements in a plant embryo-specific gene

Abstract: Previously we demonstrated the expression of a plant embryo-specific gene encoding the alpha' subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), in transgenic petunia plants. To examine the regulatory elements that control the expression of this embryo-specific gene (Gmg17.1), a series of deletion mutants was made that contain the alpha'-subunit gene flanked in the 5' direction from +14 nucleotides to -8.5 kilobases (kb) relative to the site of transcription initiation. Each of these deletion mutants was introduced into the genome of petunia cells with the help of Ti-plasmid-derived vectors. Petunia plants were regenerated from transformed cells and expression of the introduced soybean gene was examined. When the alpha'-subunit gene was flanked by 159 nucleotides upstream (Gmg17.1 delta-159), the gene was expressed at a low level in immature embryos. When the gene was flanked by 257 nucleotides upstream of the site of transcription initiation (Gmg17.1 delta-257), a high level of expression was obtained. An additional 8 kb of DNA sequence (which includes the sequence GTGGATAG at -560, which is identical to the core enhancer sequence of simian virus 40 and some animal genes) did not significantly increase the level of expression. The increase in expression level between the delta-159 and delta-257 mutants was at least 20-fold. Analysis of the nucleotides between delta-159 and delta-257 reveals four repeats of a 6-base-pair (G + C)-rich sequence (see formula in text). The deletion Gmg17.1 delta-159 contains a single AACCCA sequence. We suggest that the (G + C)-rich repeats play a critical role in determining the level of expression of the transgenic plants.

Photosynthesis-Associated Gene Families: Differences in Response to Tissue-Specific and Environmental Factors

Abstract: The endogenous small subunit of the ribulose-1,5-bisphosphate carboxylase gene rbcS and the light-harvesting chlorophyll a/b-binding protein gene (LHCP) of pea are expressed in a light-inducible manner and are active mainly in green chloroplast-containing tissue. Chimeric genes under control of the 5'-flanking sequences of the rbcS ss3.6 or LHCP AB80 genes from pea were used to study the factors relating to the issue-specific and lightinducible expression of these nuclear-encoded genes in transgenic tobacco plants. The results show that plastid development plays a crucial role in the activation of expression of these chimeric genes. Particular members of each of the above gene families respond differently to tissue-specific and environmental factors. Furthermore, the light-inducible expression directed by the 5'-flanking sequence of ss3.6 rbcSgene is not exclusively mediated by phytochrome, but probably is controlledin large part by another photoreceptor.

Transgenic mice with mu and kappa genes encoding antiphosphorylcholine antibodies.

Abstract: Transgenic mice were produced that carried in their germlines rearranged kappa and/or mu genes with V kappa and VH regions from the myeloma MOPC-167 kappa and H genes, which encode anti-PC antibody. The mu genes contain either a complete gene, including the membrane terminus (mu genes), or genes in which this terminus is deleted and only the secreted terminus remains (mu delta mem genes). The mu gene without membrane terminus is expressed at as high a level as the mu gene with the complete 3' end, suggesting that this terminus is not required for chromatin activation of the mu locus or for stability of the mRNA. The transgenes are expressed only in lymphoid organs. In contrast to our previous studies with MOPC-21 kappa transgenic mice, the mu transgene is transcribed in T lymphocytes as well as B lymphocytes. Thymocytes from mu and kappa mu transgenic mice display elevated levels of M-167 mu RNA and do not show elevated levels of kappa RNA, even though higher than normal levels of M-167 kappa RNA are detected in the spleen of these mice. Approximately 60% of thymocytes of mu transgenic mice produce cytoplasmic mu protein. However, despite a large amount of mu RNA of the membrane form, mu protein cannot be detected on the surface of T cells, perhaps because it cannot associate with T cell receptor alpha or beta chains. Mice with the complete mu transgene produce not only the mu transgenic mRNA but also considerably increased amounts of kappa RNA encoded by endogenous MOPC-167 like kappa genes. This suggests that B cells are selected by antigen (PC) if they coexpress the mu transgene and appropriate anti-PC endogenous kappa genes. Mice with the mu delta mem gene, however, do not express detectable levels of the endogenous MOPC-167 kappa mRNA. Like the complete mu transgene, the M-167 kappa transgene also causes amplification of endogenous MOPC-167 related immunoglobulins; mice with the kappa transgene have increased amounts of endogenous MOPC-167-like mu or alpha or gamma in the spleen, all of the secreted form. Implications for the regulation of immunoglobulin gene expression and B cell triggering are discussed.

Tissue-specific and ectopic expression of genes introduced into transgenic mice by retroviruses.

Abstract: Recombinant retroviruses containing the complete genomic human beta globin gene (under the control of its own promoter) and the bacterial neomycin phosphotransferase gene (under the control of the normal or enhancerless viral promoter) were used to derive transgenic mouse strains by infection of preimplantation embryos. Expression of the beta globin gene in hematopoietic tissues was observed in all transgenic strains. In addition, one strain showed ectopic expression of beta globin in the same tissues that also expressed high levels of RNA from the viral promoter. It is likely that expression from the long terminal repeat (LTR), in contrast to expression from the internal promoter, is dependent on the site of integration. Thus, retroviral vectors can be used for tissue-specific expression of foreign genes in transgenic mice, as well as for the identification of loci that allow developmental activation of a provirus.

T-DNA structure in transgenic tobacco plants with multiple independent integration sites

Abstract: Transgenic tobacco plants were produced by inoculation of leaf disks withAgrobacterium tumefaciens harboring a disarmed binary vector containing soybean leghemoglobin Lbc3 and glycinin G2 genes. Physical and genetic characterization of these plants indicated that one to six copies of DNA from the vector were transferred and maintained in the plant genome. Approximately 30% of the copies transferred were found to be incomplete or rearranged and in some cases joined as inverted repeats. The transferred DNA was found at multiple genetic loci in five of the six cases examined. In one plant, kanamycin-resistance traits were at four independent chromosomal positions, although two were genetically linked at about 3 centimorgans. Thus,Agrobacterium-mediated DNA transfer to plants has some characteristics in common with “natural” systems in animals, such as retroviral or P-element derived systems, some characteristics in common with “artificial” systems, such as microinjection, electroporation, or calcium phosphate coprecipitation techniques, and some novel characteristics.

Regulated expression of a murine class I gene in transgenic mice.

Abstract: The major histocompatibility complex class I genes play an essential role in the immune presentation of aberrant cells. To gain further insight into the regulation of the expression of these class I genes and to better define the functions of their protein products, we made use of the technique of gene transfer into the germ line of inbred mice. With the use of locus-specific DNA probes, we observed that a transgenic class I gene was expressed in a tissue-dependent fashion analogous to that of an endogenous class I gene. In addition, the level of expression of the transgenic gene was substantially higher that that of the endogenous gene. The availability of transgenic mice properly expressing a foreign murine class I gene provides a unique system to further define the role of the class I antigens in the maturation of the immune response and in determining the malignant and metastatic phenotypes of tumor cells.

Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene.

Abstract: A full-length cDNA coding for a mutant dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3), cloned from a mouse fibroblast cell line grown in high concentrations of methotrexate (MTX), was microinjected into mouse embryos to produce transgenic mice. The DHFR cDNA product is 270-fold more resistant to MTX than the wild-type enzyme. Seventeen transgenic mouse lines, identified by Southern blotting of tail or spleen DNA, carried between 1 and 400 copies of the foreign gene per cell. Eight lines have thus far been tested for resistance to MTX. Control mice were treated until death; MTX was withdrawn from transgenic mice when a cumulative MTX dose uniformly fatal for controls was reached. The major site of MTX toxicity was the gastrointestinal tract, with death of controls resulting from fluid and weight loss. Transgenic animals were relatively resistant to these symptoms and tolerated significantly more MTX than control animals. These results show that genes conferring resistance to chemotherapeutic agents can, after transfer into intact organisms, produce systemic drug resistance.

Estrogen regulation of the avian transferrin gene in transgenic mice.

Abstract: The intact chicken transferrin gene was microinjected into fertilized mouse eggs, and the resulting transgenic animals were used to produce lines of mice containing integrated copies of the chicken gene. The levels of expression of the chicken gene were quantitated in various tissues, and the response of the gene to estrogen stimulation was measured after chronic or acute estrogen exposure. Two of the three mouse lines studied maintained stable levels of expression in successive generations of offspring, and the third line had two- to threefold-higher levels in offspring than in the original parent. In the third line, the original transgenic parent was found to be a mosaic. The chicken transferrin gene was expressed at 10- to 20-fold-higher levels in liver than in any other tissue; however, the levels of chicken transferrin mRNA in kidney were higher than expected, indicating that the tissue specificity was only partial. In all three lines, the foreign gene was induced by estrogen administration. After 10 days of estrogen administration, there was a twofold increase in both transferrin mRNA and transcription of the chicken transferrin gene. A single injection of estradiol led to a fourfold increase in transferrin mRNA synthesis at 4h. As a control the levels of mouse albumin were measured, and both the level of albumin mRNA and its rate of transcription declined about twofold after estrogen administration. Our results indicate that the intact chicken gene with 2.2 kilobases of 5' flanking sequence contains signals for both tissue specificity and steroid regulation that can be recognized in mice.

T cell independent Thy-1 allo-antibody response with the use of transgenic mice.

Abstract: We have introduced a mouse Thy-1.1 gene into the germline of Thy-1.2 mice. The introduced gene was shown to be expressed at very high levels in thymocytes when compared with the endogenous gene. Transgenic thymocytes were shown to evoke a higher than normal primary anti-Thy-1.1 antibody response in plaque-forming cell (PFC) assays. This result suggests that a direct quantitative interaction of the Thy-1 antigen activates the B cell response.

Rearrangements of microinjected recombinant DNA in the genome of transgenic mice.

Abstract: Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the “plasmid rescue” technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.

TRANSGENIC MICE WITH j,AND K GENES ENCODING ANTI PHOSPHORYLCHOLINE ANTIBODIES

Abstract: Analysis of the antibody response on the cellular and molecular level is complicated by the fact that B lymphocytes are an enormously heterogenous population with respect to the immunoglobulin genes they express. It has been possible to alleviate this obstacle by studying monoclonal populations of myeloma cells or by immortalizing individual B cells in hybridomas . However, these cells are generally arrested in a particular stage of differentiation and do not permit the study of the dynamics of cell development and interaction. The introduction of rearranged Ig genes into the germline of mice has been a method to study a monoclonal response on the level of the whole animal (1-3) . Transgenic mice have provided a unique and powerful tool to analyze the expression of Ig genes. Transgenic mice are produced by microinjection of cloned genes into the male pronucleus of fertilized eggs, and implantation of the embryos into the uterus of a foster female (4) . We have previously produced transgenic mice with the functional K gene from the myeloma MOPC-21 (1) . We found that the expression of this rearranged K transgene is restricted to B lymphocytes (5, 6), and that coexistence in a B cell of transgenic a and endogenous H chains prevents rearrangement of endogenous K genes (7, 8) . Apparently, allelic exclusion of K genes is regulated by a feedback from a complete Ig molecule, not by free L chains . It was important to check these findings with another K gene that contains a different V region and 5' upstream sequences . Also, the MOPC-21 K chain previously used cannot be secreted alone. The possibility of feedback by K chains that can be secreted on their own needs to be evaluated . Furthermore, it has been reported (3, 9) that heavy chain transgenes cause feedback inhibition of H gene rearrangement. It will be important to determine whether this finding can be generalized by using H transgenes with a different V region . Beyond simply establishing the fact that H genes, or their products, and H plus L chains cause feedback inhibition of H and K gene rearrangement, the molecular mechanism will have to be addressed. It appears possible that insertion of the H chain into

Transhybridomas from fusion gene transgenic lymphocytes can be used to produce foreign protein of biological interest

Abstract: A method is described for achieving the efficient production of proteins of biological interest by the establishment of hybridomas from lymphocytes of transgenic animals carrying a fusion gene having promoter-regulatory sequences functional in lymphocytes fused to the coding sequences of the protein to be produced. While possible improvements in the method are discussed, it is anticipated that the method will ultimately be applicable to the production of any protein of interest.

Transgenic Mouse Preparation

Abstract: This chapter discusses the preparation of the transgenic mouse. Transgenic mice are generated by the introduction of a cloned piece of DNA into a fertilized mouse egg, with subsequent growth of the embryo to birth and beyond. This method allows the function of specific genes and regulatory elements to be examined in the context of the whole animal with its complex program of development and tissue differentiation. This method involves the removal of freshly fertilized eggs from a female mouse. These embryos are washed and prepared for injection. Embryos are individually immobilized by a holding micropipette while cloned DNA pieces are injected into the male pronucleus through a second injection pipette. Following injection, groups of embryos are placed in the uterine tubes of pseudopregnant female mice. By coinserting regulatory sequences with the coding region of interest, expression of the gene is regulated. Other transgenic insertions serve to disrupt the host's genes and lead to functional alterations. It is likely that each embryo injected will have the same piece of DNA inserted at random locations in the genome. The resulting births are screened for the presence of the cloned DNA within the genome.

Gene transfer by direct pronuclei microinjection.

Abstract: The technique of micromanipulating and injecting a mouse egg was first described in 1966 by T.P. Lin (8,9). With the advent of recombinant DNA technology, it has become possible to transform cultured cells by modifying this technique to microinject directly into the cell nuclei-specific cloned gene sequences (1,4). This approach is amenable only to established cell lines in culture, and so its general usefulness in the analysis of tissue-specific gene expression has been found to be somewhat limited. Subse-quently, transgenic animals have been produced by introduction of foreign genes at the pronuclear stage of fertilized, one-cell zygotes. Most of the successes have been with mouse eggs (3,5,6,11,12), and only recently has the successful production of transgenic animals been extended to the rabbit, pig, and sheep (7). This technique has become a powerful tool for studying gene regulation and physiological functions of gene products in a normal host environment. This chapter summarizes the current methodology used for mouse eggs in gene transfer experiments by direct pronuclear microinjection.