Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.     

Regulation of human insulin gene expression in transgenic mice

Insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1,2). It is synthesized by the beta-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. Insulin consists of two polypeptide chains (A and B, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (C-peptide). Humans, like most vertebrates, contain one proinsulin gene, although several species, including mice and rats, have two highly homologous insulin genes. We have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. We report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent.     

Site-independent expression of the chicken beta A-globin gene in transgenic mice

The level of expression of exogenous genes carried by transgenic mice typically varies from mouse to mouse and can be quite low. This behaviour is attributed to the influence of the mouse chromatin near the site of transgene integration. This 'position effect' has been seen in transgenic mice carrying the human beta-globin gene. It was however, abolished when DNase I hypersensitive sites (normally found 65 to 44 kilobases (kb) upstream) were linked to the human beta-globin transgene. Thus, the upstream DNA (previously named a dominant control or locus activation region, now denoted a locus control region) conferred the ability to express human beta-globin at high levels dependent on copy number on every mouse carrying the construct. We report here an investigation of chicken beta A-globin gene expression in transgenic mice. A 4.5-kb fragment carrying the beta A-globin gene and its downstream enhancer, without any far upstream elements, is sufficient to ensure that every transgenic mouse expresses chicken globin messenger RNA at levels proportional to the transgene copy number. Thus the chicken DNA elements that allow position-independent expression can function in mice. In marked contrast to the human beta cluster, these elements are no farther than 2 kb from the gene. The location of the elements within the cluster demonstrates that position independence can be mediated by DNA that does not define a gene cluster boundary.     

Tissue-specific expression of rat myosin light-chain 2 gene in transgenic mice

One approach to determining how the differential expression of specific genes is regulated in higher organisms is to introduce cloned copies of the genes (or parts of the genes) into the genomes of individual organisms from the very beginning of their development. The way in which the exogenous genetic information behaves during the development of the experimental organisms can then provide a means of defining the DNA sequences that restrict the expression of the gene to specific cell types and times of development. So far, several different genes have been introduced into the genomes of mice, but in only a few cases have the exogenous genes retained the tissue specificity of expression of the equivalent endogenous genes. I report here that in two out of three 'transgenic' mice carrying copies of the rat gene for skeletal muscle myosin light chain 2, the exogenous gene is expressed specifically in skeletal muscle cells. The sequences contained in the cloned copy of the myosin light-chain 2 gene used in these experiments are thus sufficient to confer a tissue-specific pattern of expression.     

Production of transgenic blastocyst of sheep by somatic cell cloning

Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP- N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P＞0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.     

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E

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.     

Production of transgenic rabbits, sheep and pigs by microinjection

Direct microinjection has been used to introduce foreign DNA into a number of terminally differentiated cell types as well as embryos of several species including sea urchin, Candida elegans, Xenopus, Drosophila and mice. Various genes have been successfully introduced into mice including constructs consisting of the mouse metallothionein-I (MT) promoter/regulator region fused to either the rat or human growth hormone (hGH) structural genes. Transgenic mice harbouring such genes commonly exhibit high, metal-inducible levels of the fusion messenger RNA in several organs, substantial quantities of the foreign growth hormone in serum and enhanced growth. In addition, the gene is stably incorporated into the germ line, making the phenotype heritable. Because of the scientific importance and potential economic value of transgenic livestock containing foreign genes, we initiated studies on large animals by microinjecting the fusion gene, MT-hGH, into the pronuclei or nuclei of eggs from superovulated rabbits, sheep and pigs. We report here integration of the gene in all three species and expression of the gene in transgenic rabbits and pigs.     

Deregulated c-fos expression interferes with normal bone development in transgenic mice

Transgenic mice were generated expressing c-fos genes under the control of the human metallothionein promoter. Although high levels of c-fos messenger RNA were detectable in a variety of tissues, deregulated c-fos expression specifically interfered with normal bone development without inducing malignant tumours.     

Correction of RNA aberrant splice increases foreign gene expression in transgenic mice

Transgenic mice with mammary gland secreting human granulocyte colony stimulating factor (G-CSF) were produced using mice whey acid protein gene promoter. It was found that there was very low expression level in mammary gland. Human G-CSF cDNA was obtained by RT-PCR from transgenic mice mammary gland. Sequence analysis showed that this G-CSF gene deleted the 4th exon, and compared with human G-CSF genomic DNA, there were donor and acceptor splice sites in the deletion fragment. It was considered that the 3rd and 4th introns also delete in G-CSF fragment. The transgenic construct was corrected by deleting the 3rd and 4th introns to construct the minigene, which was used to produce transgenic mice by microinjection. Northern blot showed that G-CSF expression using the new construct increased 5.4 times as that before in transgenic mice. The results suggested that it was possible that RNA aberrant splice result in low expression in transgenic mice.     

Specific expression of a foreign beta-globin gene in erythroid cells of transgenic mice

The globin gene family represents an attractive system for the study of gene regulation during mammalian development, as its expression is subject to both tissue-specific and temporal regulation. While many aspects of globin gene structure and expression have been described extensively, relatively little is known about the cis-acting DNA sequences involved in the developmental regulation of globin gene expression. To begin to experimentally define these regulatory sequences, we have taken the approach of introducing cloned globin genes into the mouse germ line and examining their expression in the resulting transgenic animals. Here we describe a series of transgenic mice carrying a hybrid mouse/human adult beta-globin gene, several of which express the gene exclusively or predominantly in erythroid tissues. These studies demonstrate that regulatory sequences closely linked to the beta-globin gene are sufficient to specify a correct pattern of tissue-specific expression in a developing mouse, when the gene is integrated at a subset of foreign chromosomal positions.     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice

Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.     

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.     

An embryonic pattern of expression of a human fetal globin gene in transgenic mice

During the evolution of the beta-globin family gene in vertebrates, different globin genes acquired different developmental patterns of expression. In mammals, specific 'embryonic' beta-like globins are synthesized in the earliest erythroid cells, which differentiate in the yolk sac of the embryo. In most mammals the embryonic globin chains are replaced by 'adult' beta-globins in fetal and adult erythrocytes, which arise in the liver and bone marrow, respectively. However, in simian primates (including humans), a distinct 'fetal' type of beta-like globin chain predominates in fetal erythroid cells. Based on the pattern of DNA sequence homologies between different mammalian species, these fetal globin genes, G gamma and A gamma, are thought to have descended from an ancestral gene, 'proto-gamma', which was embryonic in its pattern of expression. In the mouse, as well as in most other mammalian species, the descendants of the proto-gamma gene continue to function as embryonic genes. To investigate the evolutionary changes that led to the 'fetal recruitment' of the gamma-globin genes in primates, we have introduced the cloned human G gamma-globin gene into the mouse germ line. We report here that the human G gamma gene reverts to an embryonic pattern of expression in the developing mouse. This observation suggests that during evolution a shift occurred in the timing of expression of a trans-acting signal controlling the proto-gamma gene.     

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.     

Dysmyelination in transgenic mice resulting from expression of class I histocompatibility molecules in oligodendrocytes.

Major histocompatibility complex (MHC) molecules are not normally expressed in the central nervous system (CNS). However, aberrant expression has been observed in multiple sclerosis lesions and could contribute to the destruction of myelin or the myelinating cells known as oligodendrocytes. The mechanism of cell damage associated with aberrant MHC molecule expression is unclear: for example, overexpression of class I and class II MHC molecules in pancreatic beta cells in transgenic mice leads to nonimmune destruction of the cells and insulin-dependent diabetes mellitus. We have generated transgenic mice that express class I H-2Kb MHC molecules, under the control of the myelin basic protein promoter, specifically in oligodendrocytes. Homozygous transgenic mice have a shivering phenotype, develop tonic seizures and die at 15-22 days. This phenotype, which we term 'wonky', is due to hypomyelination in the CNS, and not to involvement of the immune system. The primary defect appears to be a shortage of myelinating oligodendrocytes resulting from overexpression of the class I MHC molecules.     

Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI

Epidemiological surveys have identified a strong inverse relationship between the amount in the plasma of high density lipoproteins (HDL), apolipoprotein AI (ApoA-I), the major protein component of HDL, and the risk for atherosclerosis in humans. It is not known if this relationship arises from a direct antiatherogenic effect of these plasma components or if it is the result of other factors also associated with increases in ApoA-I and HDL levels. Because some strains of mice are susceptible to diet-induced formation of preatherosclerotic fatty streak lesions, and because of available techniques for the genetic manipulation of this organism, the murine system offers a unique setting in which to investigate the process of early atherogenesis. To test the hypothesis that induction of a high plasma concentration of ApoA-I and HDL would inhibit this process, we studied the effects of atherogenic diets on transgenic mice expressing high amounts of human ApoA-I. We report that transgenic mice with high plasma ApoA-I and HDL levels were significantly protected from the development of fatty streak lesions.     

Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice

The major problem in the study of T-cell development is that of tracking thymocytes of a given specificity. Recent studies have exploited natural correlations between the expression of a particular V beta gene segment and T-cell receptor (TCR) specificity. We and others (refs 5, 6 and M. Davis, personal communication) have taken an alternative approach. We have generated transgenic mice expressing the alpha beta antigen receptor from the cytotoxic T-lymphocyte clone 2C (ref. 7). In transgenic mice of the same haplotype as the 2C clone, the 2C TCR was expressed on 20-95% of peripheral T cells. Very few of these T cells carried the CD4 antigen; the vast majority were CD4-CD8+ and were able to lyse targets with the same specificity as the original 2C clone. These results indicate that the alpha beta heterodimer transfers specificity to recipient cells as expected from earlier studies, and that receptor specificity in T-cell repertoire selection is determined by both alpha beta heterodimer and CD4 or CD8 accessory molecules.