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Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.

A plasmid was constructed with the promoter/regulatory region of the mouse metallothionein-I gene fused to the structural gene of herpesvirus thymidine kinase. When mouse eggs were microinjected with this plasmid and incubated with cadmium (a natural inducer of metallothionein gene transcription) thymidine kinase activity increased approximately 10-fold compared with control eggs not exposed to cadmium. Analysis of a set of deletion mutants revealed that the minimum sequence required for cadmium regulation lies within 90 nucleotides of the transcription start site.

Immunoglobulin genes are normally expressed only in cells of the B lymphocyte lineage after a variable (V) and constant (C) gene rearrangement has occurred. To study the control of immunoglobulin gene expression in a defined situation, we have produced transgenic mice by microinjecting a rearranged mouse immunoglobulin kappa gene (designated pB1-14) into fertilized mouse eggs. We present here the analysis of six different kappa-transgenic mouse lines. All the transgenic mice express the microinjected kappa gene in a completely tissue-specific fashion. Transcripts from pB1-14 are found at a high level in the spleen, but are undetectable in nonlymphoid tissues of testis, liver, kidney, heart, muscle, brain and thyroid gland. In lymphoid cell subpopulations, the level of pB1-14 transcripts is correlated with the relative number of B cells; there is no correlation with the proportion of T lymphocytes. We concluded, therefore, that the microinjected kappa gene contains target sequences for B lymphocyte-specific gene activation signals that override the influence of the integration site.

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigenes by the strict criteria that such antigenes stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.

Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process1–9, because spontaneous mutation rates observed for other genes are extremely low10. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites6,8, from the absence of mutations in unrearranged V genes4,5,11 and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles12. Another model in which mutations accumulate with each cell division, rather than being introduced all at once8, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations13–17. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged K-transgenes. We found that these K-transgenes were also somatically mutated. This and other observations indicated that: (1) ongoing rearrangement is not required for mutation; (2) there are signals for hypermutation in the trans-genes; (3) the mutations are found only in the variable region, so the constant region may not be a target; (4) different transgene insertion sites are compatible with hypermutations and (5) more than one transgene is expressed in the same cell.

A DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone was microinjected into the pronuclei of fertilized mouse eggs. Of 21 mice that developed from these eggs, seven carried the fusion gene and six of these grew significantly larger than their littermates. Several of these transgenic mice had extraordinarily high levels of the fusion mRNA in their liver and growth hormone in their serum. This approach has implications for studying the biological effects of growth hormone, as a way to accelerate animal growth, as a model for gigantism, as a means of correcting genetic disease, and as a method of farming valuable gene products.

THE repertoire of receptors expressed by peripheral T cells is the result of two selective events that occur during intrathymic development. Positive selection expands cells able to recognize foreign peptides presented by self MHC molecules, and negative selection eliminates cells reactive to self MHC molecules and associated self peptides1–4. Chimaera studies suggest that, at least in the case of T cells recognizing MHC class II, interaction with thymic cortical epithelial cells is responsible for the former5, whereas thymic medullary cells, of bone marrow origin, mediate the latter3, 6–8. This view of thymic development is supported by recent morphometric analyses9, showing that autoreactive cells are found in thymic cortex but not medulla. Although numerous1–3, 10–12 studies have shown that MHC class II molecules are expressed in both sites, none provides any explanation for the differential selection of T cells that is observed. Here, we describe a novel MHC class II epitope which is found on cells in thymic medulla but not cortex. The antibody to this epitope reacts with about 10% of class II molecules on B cells and may be recognizing a self peptide–MHC complex. These results provide the first evidence for differential expression of class II epitopes in different tissues and are compatible with the hypothesis that different ligands2, 13–16, rather than different affinity thresholds for the same ligand17, 18, are involved in positive and negative selection of the T-cell repertoire.

The mouse homoeobox-containing genes exhibit temporally and spatially specific patterns of expression in embryonic and adult tissues and are thought to be important in regulation of development and cellular differentiation, perhaps by mechanisms analogous to homoeotic genes in Drosophila melanogaster1–4. There has been no direct demonstration that expression of these mammalian genes can affect developmental processes, however. Hox-1.4, like other mouse homoeobox-containing genes, has been shown to be expressed in specific regions of the mid-gestation embryo5–8, but is unique in that its highest level of expression in the adult animal is restricted to developing male germ cells5,8,9. We have introduced a construct carrying the mouse Hox-1.4 gene into the germ line of mice to begin to identify the m-acting elements required for proper expression and to assess the consequences of increasing Hox-1.4 gene expression. The construct was designed to produce normal Hox-1.4 protein from transcripts that are distinguishable from the products of the endogenous gene. The integrated transgene seemed to exhibit the appropriate tissue specificity of expression, but transcript levels were elevated in certain tissues, particularly the embryonic gut. This overexpression correlated with changes in the normal developmental program of the gut, resulting in an inherited abnormal phenotype known as megacolon.

There has been suggestive evidence for embryonic genome activity in the preimplantation mouse embryo for several years. Whitten and Dagg (1961) found a paternal effect on cleavage rate before implantation, and Morris (1968) suggested that the X chromosome is necessary for early cleavage. However, to prove the presence of paternally derived gene activity, it is necessary to demonstrate the existence of a protein in the embryo coded for by a paternal allele. Chapman et al. (1971) have recently shown the presence on day 5 of development of the paternal variant of glucose phosphate isomerase (GPI) in mouse embryos derived by mating males and females containing the genes for different isozyme subunits. In addition, the work of Epstein (1972) on the total activity of hypoxanthine-guanine phosphoribosyl transferase suggests that the paternal allele for this enzyme, which is sex linked, is active at day 4 in development. Work in our laboratory during the past 2 years has also identified the paternal variant of GPI in the mouse embryo but at an earlier stage of development than previously reported. The studies reported here demonstrate paternal allele activity of GPI on the third day of development after only three cleavage divisions. This is not only earlier than previously reported for the mouse but is much earlier than found in fish, frog, and quail.

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.

It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell1. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species2–4. This system has proved useful in understanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte5,6. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell7,8. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system.

The dwarf little (lit) mouse is a model for the human hereditary disorder, isolated growth hormone (GH) deficiency type I. In these animals, dwarfism results from an autosomal recessively inherited gene mutation. The GH gene is present but production of GH mRNA is deficient, resulting in reduced serum GH and concomitantly decreased serum somatomedin. Growth retardation is evident by 15 days of age and adult animals reach approximately one-half normal size. Mutant mice of both sexes also exhibit a delayed onset of puberty, with males having a high degree of infertility. As administration of GH restores growth, we reasoned that growth failure in the mutant mice might be corrected by providing them with sufficient GH by gene therapy. Here we demonstrate that although the rat and human GH genes alone do not restore growth in transgenic mutants, a metallothionein-rat growth hormone fusion gene (MT-rGH) does. Moreover, the fertility of transgenic mutant males is improved; however, female fertility is impaired.

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of inmiunoglobulin1–3, insulin and chymotrypsin4 genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunglobulin κ gene is expressed specifically in B lymphocytes5,6, a rat elastase I gene is expressed specifically in pancreas7 and a chicken transferrin gene is expressed preferentially in liver8. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes9, but initial experiments with β-globin genes have not revealed proper regulation10–12. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5′-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.

Recent experiments have shown that the microinjected kappa-chain gene of transgenic mice is expressed in a tissue-specific fashion only in B lymphocytes. The next step was to determine whether, within the B-lymphocyte lineage, the kappa-chain gene was expressed in a normal developmental fashion. Normally, only mu heavy(H)-chain genes, and not kappa-chain genes, are expressed in pre-B cells. To obtain cloned cell lines derived from early cells of the B-cell lineage, we transformed bone marrow cells from kappa-transgenic mice with Abelson murine leukaemia virus (A-MuLV) and tested the resultant cell lines for the retention of the kappa transgene and its expression in RNA and protein. We found that cells with the pre-B phenotype exist in kappa-transgenic mice. We further observed that in A-MuLV-transformed cell lines from a kappa-transgenic mouse with a high copy number of the transgene, the proportion of cell lines expressing kappa (transgenic kappa) was higher than in cell lines from normal or low copy number transgenic mice.

UNTIL recently, studies on early mammalian embryos were relatively infrequent because of difficulties in obtaining material and in maintaining the viability of the embryos. New methods for routine collection and culture1 have partially overcome these obstacles and several specific biochemical requirements for growing mouse embryos in vitro have been determined2–6. Using these methods, we have determined the oxygen concentration required by mouse embryos developing in vitro.

ACTIVITY of lactic dehydrogenase (L-lactate: NAD+ oxidoreductase, E.C, 1.1.1.27) has been determined in the preimplantation mouse embryo1. The one-cell mouse embryo had a lactic dehydrogenase (LDH) activity nine times greater than the most active adult mouse tissue. This finding, in addition to several other observations, indicates the importance of this enzyme to the early mouse embryo and perhaps to all mammalian embryos. Culture work has shown that the two-cell mouse embryo can develop into a blastocyst in vitro when pyruvate or lactate is present in the medium as the only energy source but not when glucose alone is present2. The optimum concentration of lactate in the medium is 5 × 10−2 moles/l, compared with 5×10−4 moles/l, for pyruvate3. The concentration of lactate is large in the Fallopian tube of the rabbit during the first 3 days after ovulation4.