Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice

Transgenic mice were produced by microinjection of a rearranged, functional immunoglobulin kappa gene into fertilized mouse eggs and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were mated and the DNA, RNA and serum kappa chains of their offspring were analysed. The data from offspring of three different transgenic mice indicate that the microinjected gene is expressed in the spleen, but not the liver of mice which inherited the injected gene.     

Pre-B cells in kappa-transgenic mice

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.     

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.     

Depletion of the predominant B-cell population in immunoglobulin mu heavy-chain transgenic mice

The transgenic mouse line M54 was generated by introducing a functionally-rearranged immunoglobulin mu heavy-chain gene into the germ line of a C57B1/6 inbred mouse. Previous examination of the antibodies produced by B-cell hybridomas derived from transgenic M54 mice showed that the presence of the mu transgene grossly altered the immunoglobulin repertoire of unimmunized animals, suggesting that these mice suffer from a serious immunoregulatory perturbation. Studies presented here introduce a new perspective on this functional defect. We show that the lymphoid tissues from these transgenic mice lack virtually all conventional bone-marrow-derived B cells, which constitute the predominant B-cell population in normal mice and which typically produce primary and secondary antibody responses to T-cell-dependent antigens. Moreover, the bone marrow from transgenic M54 mice is depleted of pre-B lymphocytes, indicating a serious defect in early B-cell lymphopoiesis. In contrast, CD5 (Ly-1) B cells, a second B-cell population displaying a characteristic set of cell surface markers which are derived from distinct precursors in the peritoneum, are represented at normal frequencies in these transgenic mice. Thus, the presence of the rearranged immunoglobulin heavy-chain transgene in M54 mice results in an unexpected selective developmental defect that impairs the development of bone-marrow-derived pre-B and B cells without affecting Ly-1 B cells.     

Transmission and expression of a specific pair of rearranged immunoglobulin mu and kappa genes in a transgenic mouse line

Two genes that code for a hapten-specific immunoglobulin M (IgM) have been introduced into the mouse germ line. The transgenic antibody represents 10-50% of the serum IgM and is expressed on the membrane of B cells. B-cell hybridoma lines show that a negative feedback inhibition of mu and kappa transgenic products on the immunoglobulin heavy-chain rearrangement is possible.     

High expression of human serum albumin in milk of trans- genie mice directed by the goat p-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5'upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

The immunoglobulin mu constant region gene is expressed in mouse thymocytes

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.     

小鼠转基因及传代研究

综述了湖北省农科院生物技术研究所近年来有关转基因小鼠的主要研究进展。应用原核注射技术 ,将POMT PGH、hDAF、pBHSA、pSHSA、PT HBV 1 3、Bcl xL、hCD5 9、hCD5 9+hMCP等 8种外源基因 ,注入并移植 4 874枚小鼠的受精卵 ,得到 5 5 6只小鼠 ;经PCR和Southern杂交检测 ,确认原代转基因小鼠 10 8只。基因整合率平均 19.4 % ,转基因效率平均 2 .2 %。应用混合注射的方法得到了转双基因小鼠 ,双基因共整合率 2 2 .2 %。表达外源蛋白的转基因小鼠在 5 0 %～ 10 0 %之间。研究了小鼠的超数排卵和影响转基因效率的几个因素。通过转Bcl xL小鼠与非转基因小鼠连续五个世代的传代交配和检测 ,研究了转基因小鼠外源基因的遗传规律 ,表明四只原代小鼠中 ,只有一只能稳定地将外源基因传递给后代。并非所有的转基因小鼠都具有遗传的稳定性 ,欲建立小鼠的转基因品系 ,尚需对原代转基因小鼠进行筛选。     

Effect of HS2 and HS3 elements on erythroid-specific expression in transgenic mice

The expression plasmids CMV/GFP, HS2ALL, HS3ALL and HS23ALL were selected to investigate the effect of HS2 and HS3 element on erythroid-specific expression in transgenic mice. These plasmids were digested with restriction enzymes and purified. And five DNA fragments, CMV/GFP, HS2/GFP, CMV/HS2/GFP, HS23/GFP and HS3/GFP were obtained. After purification, the above DNA fragments were microinjected into the pre-nuclei of the mice fertilized eggs and transgenic mice were generated, with an integration rate of 10.89%. The green fluorescence protein(GFP) expression in many transgenic mouse tissues was determined by FACS analysis. The results showed that the HS2 and 1.7 kb of β-globin gene promoter were sufficient for the erythroid-specific expression of β-globin gene. The GFP expression of different recombinant constructs was also analyzed in blood of all the transgenic mice with FACS. The results indicated that HS2 and HS3 had the same enhancement activity on the regulation of β-globin gene expression. Moreover, these two elements showed a significant synergistic effect on gene expression at the transgenic mouse level, although the GFP expression varied largely among different transgenic mouse litters.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin 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 immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. 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.     

Variant (6 ; 15) translocation in a murine plasmacytoma occurs near an immunoglobulin kappa gene but far from the myc oncogene

Chromosome translocations in B-lymphoid tumours are providing intriguing insights and puzzles regarding the role of immunoglobulin genes in the activation of the myc oncogene (reviewed in refs 1, 2). The 15 ; 12 translocations found in most murine plasmacytomas and the analogous 8 ; 14 translocation in human Burkitt's lymphomas involve scissions of murine chromosome 15 (human chromosome 8) near the 5' end of the c-myc gene and subsequent fusion near an immunoglobulin heavy-chain gene. The less well characterized 'variant' translocations found in about 15% of such tumours also involve the myc-bearing chromosome band, but exchange occurs with a chromosome bearing an immunoglobulin light-chain locus--in mice, the kappa-chain locus bearing chromosome 6 (refs 3-5) and, in man, chromosome 2 (or 22), at the same band at which the kappa (or lambda) locus lies (reviewed in ref. 1). The Burkitt variant translocations involve scissions 3' of c-myc; one 8 ; 22 translocation placed the C lambda locus just 3' of c-myc, but usually the chromosome 8 breakpoint is a greater, but unknown, distance away from c-myc, more than 20 kilobases (kb) in one 8 ; 2 translocation involving the C kappa gene. Little is known about the murine 6 ; 15 translocations, although a C kappa gene cloned from one plasmacytoma (PC7183) is linked, via chromosome 12 sequences, to an unidentified region of chromosome 15 (ref. 11). We describe here the chromosome fusion region from plasmacytoma ABPC4, which displays the typical reciprocal 6;15 translocations. We find that the chromosome 6 breakpoint is near C kappa but, unlike those in the heavy-chain locus, not at a position where immunoglobulin genes normally recombine. Moreover, the chromosome 15 sequences involved in the ABPC4 translocation are not derived from the vicinity of c-myc.     

Vill转基因小鼠的制备

摘要:目的　通过制备Vill转基因小鼠研究该基因的功能。 方法与结果　首先由RT-PRC方法获得全长约2605bp的Vill基因;经T载体克隆测序验证后,以克隆载体pMD19-T-Vill为模板,设计引物并引入酶切位点,将PRC扩增产物与pEF6/V5-His-LacZ同时进行酶切、连接,构建表达载体pEF6/V5His-Vill;经真核表达验证后,酶切获得含Vill基因的显微注射DNA构件;显微注射390枚受精卵后,在出生存活的77只仔鼠中获得转基因阳性GO代小鼠19只,其中16只能够稳定遗传并建系,转基因阳性小鼠外观未有明显改变。 结论　Vill转基因小鼠为该基因的功能研究准备了实验材料。     

Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice.

Studies on transgenic mice expressing immunoglobulins against self-antigens have shown that self-tolerance is maintained by active elimination (clonal deletion), functional inactivation (clonal anergy) of self-reactive B cells, or a combination of both. We have established and characterized a transgenic mouse line expressing an anti-erythrocyte autoantibody. In contrast to other autoantibody transgenic lines, about 50% of the animals of this transgenic line suffer from autoimmune disease, indicating a loss of self-tolerance. Here we show that peritoneal Ly-1 B cells (also known as B-1 cells) are responsible for this autoimmune disease in our transgenic mice. A few self-reactive Ly-1 B cells that have somehow escaped the deletion mechanism expand in the peritoneum because of the absence of self-antigen. These Ly-1 B cells are eliminated in vivo by apoptosis once exposed to self-antigen. On the basis of these results we propose a novel autoantibody production mechanism whereby self-reactive B cells sequestered in compartments free of self-antigens may survive, proliferate and be activated for generation of pathogenic autoantibodies in autoimmune diseases.     

A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element

Several functionally important genetic elements (such as the TATA box, mRNA splice sequences, poly(A) addition signal) were first detected as short segments of unexplained sequence homology within non-coding regions of different genes. A short region of unknown sequence in the intron between the human J kappa and C kappa immunoglobulin coding regions was found to be sufficiently homologous to the corresponding segment of the mouse gene to form stable heteroduplexes. Although no specific function has yet been definitely ascribed to this region (which we call the kappa intron conserved region, or KICR), some functional significance has been inferred from the findings that (1) activation of B lymphocytes induces a DNase hypersensitivity site in this region and (2) deletions including this region reduce expression of kappa genes introduced into lymphoid cells. To delineate the KICR more precisely and to test the generality of the sequence conservation in a third species, we have sequenced this region of the human and mouse genes and have examined the corresponding region of a recently cloned rabbit kappa gene. We find a segment of about 130 base pairs (bp) that shows striking conservation in all three species, demonstrating homology significantly higher than within the C kappa coding region itself. Two short sequences from the J kappa-C kappa intron that were noted by other investigators to be homologous to proposed 'enhancer' sequences both lie within the conserved region.     

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2

The putative oncogene bcl-2 is juxtaposed to the immunoglobulin heavy chain (Igh) locus by the t(14;18) chromosomal translocation typical of human follicular B-cell lymphomas. The bcl-2 gene product is not altered by the translocation, but its expression is deregulated, presumably by the Igh enhancer E mu. Constitutive bcl-2 expression seems to augment cell survival, as infection with a bcl-2 retrovirus enables certain growth factor-dependent mouse cell lines to maintain viability when deprived of factor. Furthermore, high levels of the bcl-2 product can protect human B and T lymphoblasts under stress and thereby confer a growth advantage. Mice expressing a bcl-2 transgene controlled by the Igh enhancer accumulate small non-cycling B cells which survive unusually well in vitro but do not show a propensity for spontaneous tumorigenesis. In contrast, an analogous myc transgene, designed to mimic the myc-Igh translocation product typical of Burkitt's lymphoma and rodent plasmacytoma, promotes B lymphoid cell proliferation and predisposes mice to malignancy in pre-B and B lymphoid cells. Previous experiments have suggested that bcl-2 can cooperate with deregulated myc to improve in vitro growth of pre-B and B cells. Here we describe a marked synergy between bcl-2 and myc in doubly transgenic mice. E mu-bcl-2/myc mice show hyperproliferation of pre-B and B cells and develop tumours much faster than E mu-myc mice. Suprisingly, the tumours derive from a cell with the hallmarks of a primitive haemopoietic cell, perhaps a lymphoid-committed stem cell.     

Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion.

Burnet's clonal selection theory suggests that each B lymphocyte is committed to a single antibody specificity. This is achieved by a programme of somatic rearrangements of the gene segments encoding antibody variable (V) regions, in the course of B-cell development. Evidence from immunoglobulin-transgenic mice and immunoglobulin-gene-transfected transformed pre-B cells suggest that the membrane form of the immunoglobulin heavy (H) chain of class mu (microns), expressed from a rearranged H-chain (IgH) locus, may signal allelic exclusion of the homologous IgH locus in the cell and initiation of light (L)-chain gene rearrangement in the Ig kappa loci. We report here that targeted disruption of the membrane exon of the mu chain indeed results in the loss of H-chain allelic exclusion. But, some kappa chain gene rearrangement is still observed in the absence of micron expression.