Generation Of Transgenic Mice Research Articles

Cloning of the mouse 25-hydroxyvitamin D 3-1α-hydroxylase (CYP1α) gene

A genomic clone for 25-hydroxyvitamin D 3-1α-hydroxylase (1α-hydroxylase) was isolated from a mouse embryonic stem cell P1 genomic library. It contains nine exons spanning 4.8 kb from the transcriptional start site. All the intron insertion sites are identical to that of the human 1α-hydroxylase and human vitamin D 3 25-hydroxylase genes. A polyadenylation signal AUUAAA that differs from the consensus sequence at the second residue was identified 16 bp downstream of the 3′ end of the previously reported mouse cDNA. This element is the only common natural variant described. The 3′ end of the gene was determined using a RACE technique. Three poly(A) addition sites were observed 12, 15 and 22 bases from the AUUAAA element. Such distances are in agreement with what is required for maturation of mammalian pre-mRNAs. This molecular cloning makes possible the generation of transgenic mice in order to further investigate the role and importance of the 25-hydroxyvitamin D 3-1α-hydroxylase (CYP1α).

Expression analysis of Escherichia coli lacZ reporter gene in transgenic mice

To define a gene expression mechanism, it is often advantageous to use a reporter gene and transgenic mouse. The lacZ reporter gene is particularly useful for studies of the cis-regulatory element for tissue-specific expression in transgenic mice because of the ease of the enzyme assay and visualization on sections. In this report, we describe our method for examining the cis-regulatory element in transgenic mice, including choice of the lacZ gene, generation of transgenic mice, and analysis of β-galactosidase activity. Themes: Cellular and molecular biology Topics: Staining, tracing, and imaging techniques

Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection.

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice.

The polymeric immunoglobulin receptor (pIgR), a transmembrane protein, transports dimeric IgA (dIgA) across the epithelial cells of the mucosal surfaces into the external secretions, for example milk from the mammary glands. The pIgR is consumed during the transcytosis of dIgA and is cleaved at the apical side of the epithelial cells, regardless of the binding to its ligand (dIgA), to form secretory component (SC). We hypothesize that the expression level of the endogenous murine pIgR gene in the epithelial cells is rate-limiting for the transport of dIgA across the epithelial cells into the secretions. We address this key issue by generating transgenic mice over-expressing the pIgR gene in their mammary glands in order to examine the effect on dIgA levels in the milk. Here we report on the generation of transgenic mice and analysis of the expression level of pIgR in their mammary glands. We cloned and characterized the murine pIgR gene and constructed an expression cassette bearing the pIgR gene under the control of the regulatory sequences of the bovine alpha s1-casein gene. Four transgenic lines were made, expressing the pIgR construct at RNA and protein level only in their mammary glands. The levels of the SC protein in the milk ranged from 0.1 to 2.7 mg/ml during mid-lactation. These levels are 10-270 times higher than wild-type SC levels (0.01 mg/ml).

11] Use of yeast artificial chromosomes to express genes in transgenic mice

Publisher Summary This chapter focuses on the purification of yeast artificial chromosomes (YAC) DNA for the production of transgenic mice by microinjection of fertilized oocytes and the analysis of YAC transgene structure in mice. The chapter outlines the manipulations of YACs in yeast. Generation of transgenic mice with YACs and other large molecules has proved to be a valuable system for studies of gene structure-function relationships, production of mouse models of human disease, complementation of mouse mutants, use of mice as bioreactors, and synthesis of in vivo YAC libraries. Continued refinements of current techniques and development of new protocols should encourage widespread adaptation of this strategy for the aforementioned applications. Use of whole loci as transgenes is an important improvement as the advent of murine transgenesis, one that may allow a more realistic assessment of gene regulatory mechanisms during development.

Linearization and purification of BAC DNA for the development of transgenic mice.

Bacterial artificial chromosome (BAC) vectors are increasingly used for generation of transgenic mice due to the relatively large size and the stability of their inserts compared to YACs. We have compared methods for purification and linearization of BACs, and describe an optimised protocol for preparation of high quality linear BAC DNA based on lambda terminase digestion, electroelution of linearized DNA together with simple preliminary multiplex PCR screening to detect transgenic mice. Linearized BAC DNA purified this way was successfully used for the development of transgenic mice containing 2-4 copies of the transgene.

Sperm-mediated gene transfer in mice.

Sperm-mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm-mediated DNA transfer to mouse eggs in well-established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm-mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm-mediated transfer of foreign DNA.

P-147 - Human gene of the endothelial receptor for oxidized LDL - Cloning and generation of transgenic mice

P-147 - Human gene of the endothelial receptor for oxidized LDL - Cloning and generation of transgenic mice

Incorporation of cultured embryonic cells into transgenic and chimeric, porcine fetuses.

The derivation of murine, embryonic stem cells, and their use in the generation of transgenic mice, are well-established procedures. Application of these methodologies to non-murine species, however, remains to be fully realized. Non-murine embryonic stem cells would be of considerable value to studies in comparative development, and would raise important implications for agriculture and biotechnology. Here we report the achievement of chimerism and transgenesis in the domestic pig (Sus scrofa) at a fetal stage by the embryonic stem-cell methodology, using an embryonic cell line which previously we described as having the capacity to differentiate in vitro. This entailed: transformation, by electroporation, of a cell line, PE1, with the bacterial gene for neomycin-resistance; re-introduction of a polyclonal population of neo-PE1 cells into host blastocysts, followed by transfer of reconstructed embryos into a pseudopregnant recipient; and subsequent derivation of transgenic and chimeric porcine fetuses, as determined by two independent molecular assays of fetal genomic DNA. In the first instance, chimerism was revealed in one fetus by the presence of the transgene, as detected by Southern blotting; and in the second instance, in that and another fetus by the presence of supernumerary alleles for the class II major histocompatibility locus, SLA-DQB*C, by single-strand conformation polymorphic analysis. The contribution of neo-PE1 cells to the first chimeric and transgenic fetus was approximately 25%, and to the second chimeric fetus, below the level of detection by Southern blotting (i.e. less than 10%). The results indicate that, at the time of embryo reconstitution, a proportion of neo-PE1 cells were pluripotent and of the primary ectodermal lineage.

Expression of a bovine alpha-lactalbumin transgene in alpha-lactalbumin-deficient mice can rescue lactation. In vivo relationship between bovine alpha-lactalbumin expression content and milk composition.

Lowering the lactose content of milk in vivo has two potential benefits: to develop milk products for lactose intolerant people (Delmont, 1983) and to produce a more concentrated milk, reducing milk storage volume and milking frequency without affecting the overall fat and protein yields. Lactose synthesis in the lactating mammary gland results from the interaction of α-lactalbumin (α-la) with a Golgian UDP-galactosyltransferase (EC 2.4.1.22), and it has been suggested that lactose and α-la contents are directly related (Fitzgerald et al. 1970). Evidence that α-la is the only protein responsible for the in vivo induction of lactose synthesis came recently from knock-out experiments of the relevant gene (Stinnakre et al. 1994; Stacey et al. 1995), which disrupt lactation, as lactose-deprived milk of α-la-deficient mice is highly viscous. However, although the lactose, fat and protein contents of the milk were found to be directly related to the quantity of α-la present (Stinnakre et al. 1994), only in mice homozygous for the α-la null allele was there a significant alteration of milk concentration (Stacey et al. 1995). Stacey et al. (1995) also demonstrated that human α-la could substitute for its mouse counterpart via homologous recombination.In the present study, we report the generation of transgenic mice producing only bovine α-la by back-crossing animals from four independent bovine α-la transgenic lines with α-la-deficient mice. Our work confirms that foreign α-la can substitute for the mouse protein and suggests a negative correlation between the α-la content and protein and fat concentrations in milk.

Production and analysis of transgenic mice containing yeast artificial chromosomes.

Until the advent of transgenic mouse technology, studies of mammalian gene expression and regulation were largely limited to cell lines transfected with constructs containing limited genetic information. Developmental studies were not possible since cell lines are generally locked into one ontogenic stage. Transgenic mice provided two improvements and facilitated the study of gene function in development and disease. First, transgene expression in mice allowed assessment of phenotypes. Second, gene expression could be studied throughout development and tissue-specific regulatory elements could be analyzed. However, expression of transgenes was erratic due to position effects and copy number-independent expression. In many instances these problems were due to the nature of the transgene constructs, which typically were limited in their size due to constraints on how large a DNA fragment could be isolated without degradation and introduced into the mouse. Thus, deletion of cis sequences was necessary during the design of transgenes to be injected. Many large genes with exons spanning several hundred kilobases or multigenic loci could not be used as transgenes. cDNAs were substituted for large genes or individual genes from a multigene cluster were utilized in place of an entire locus. However, expression from these constructs was subject to the effects of surrounding chromatin into which they were integrated. Some improvement resulted when other cis-acting elements, such as enhancers, introns or polyadenylation signals were included. These additional sequences assisted studies in which the main goal was to express the transgene, but these truncated constructs lacked their natural regulatory elements and thus developmental studies were not necessarily indicative of how the native gene might be regulated. Ideally, a system in which large genes or loci could be successfully used in the generation of transgenic mice might improve the utility of transgenic studies. Inclusion of distal regulatory elements within the native locus might validate developmental studies and insulate the construct from position effects. With this in mind several lab groups successfully implemented the use of large DNA constructs, in particular yeast artificial chromosomes (YACs), as transgenes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21).

A Morphologic Approach to Understanding Molecular Events During Development

Through the combined technologies ofin situhybridization and transgenic mouse analysis major advances have been made in the understanding of developmental biology. Recent advances in understanding embryogenesis have been accomplished by determining specific gene activation during development, determining the cell specificity of individual activated genes, characterizing the cis regulatory elements of the developmentally regulated genes, and disrupting the activity and functions of these genes during critical stages of development through targeted mutation.In situhybridization provides highly specific and sensitive detailed information of both the spatial and temporal pattern of endogenous gene expression during embryogenesis. The regulatory elements that determine the tissue specific and temporal related expression pattern of these embryonic genes are then identified and characterized byin situhybridization through the generation of transgenic mice which carry gene constructs with reporter genes, such as CAT, luciferase, or lac Z, linked to the flanking DNA sequences that exert either positive or negative influence over the expression of the gene in question. Finally once the expression patterns and regulatory elements have been characterized targeted ablation of the developmentally regulated gene can determine or provide important insight into function.

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.

YAC transgenesis in farm animals: Rescue of albinism in rabbits

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis-such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene-have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation.

Generation of transgenic mice from yeast artificial chromosome DNA that has been modified by gene targeting

Yeast artificial chromosome (YAC) vectors permit the cloning of up to megabase fragments of human genomic DNA in yeast ( Saccharomyces cerevisiae). Efficient recombination between homologous segments of DNA is one of the hallmark genetic features of yeast. This characteristic facilitates the introduction of specific mutations into YACs by gene targeting. Gene targeting has been used recently to introduce specific mutations into YACs spanning the human β-globin locus and the human apolipoprotein (apo)-B gene, and the mutated YAC DNA has been used to generate transgenic mice. This approach has been useful for the study of the regulatory elements controlling β-globin gene expression and for the study of apo-B structure and function. This article reviews the techniques for introducing mutations into YACs, for the purpose of expression in transgenic mice.

Transgenic models of breast cancer metastasis.

Traditionally it has been difficult to genetically define the events that lead to the formation of metastatic mammary tumors. While a number of gene products have been implicated in the development of mammary carcinomas, few have been demonstrated to play causative roles in mammary tumor formation. With the advent of transgenic mouse technology, the basic researcher can now target the expression of a gene product to a particular tissue. This has provided scientists with the ability to create mouse models of human diseases. Of particular interest is the generation of transgenic mice carrying genes thought to play important roles in the initiation/progression of mammary carcinomas. From these model systems have emerged murine tumors that not only mimic human pathologies but have the propensity to metastasize to the lung, mirroring one of the major sites of human metastases. Here we review transgenic models of mammary tumorigenesis, with particular emphasis on those that develop metastatic mammary carcinomas.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

A transgenic mouse assay for agouti protein activity.

The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human beta-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a16H allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a16H phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein.

Visualization of Large DNA Molecules by Electron Microscopy with Polyamines: Application to the Analysis of Yeast Endogenous and Artificial Chromosomes

Standard visualization of nucleic acids by electron microscopy requires the use of special spreading techniques. The most common method takes advantage of the formation of a complex between negatively charged nucleic acid molecules and a positively charged monolayer film of proteins or cationic agents. Here, we describe an alternative protocol for the rapid visualization of DNA by electron microscopy based on the complexes formed when nucleic acids are exposed to buffers containing polyamines in the presence of sodium chloride. This procedure has been devised for the detection and analysis of large DNA molecules, such as yeast artificial chromosomes, but can be applied to DNA molecules of small size as well. The formation of DNA-polyamine complexes stabilizes large DNA molecules in solution and prevents shearing. This property allows large DNA molecules to remain intact after passage through microcapillaries used in the generation of transgenic mice by microinjection of fertilized eggs.

Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs.

Vertebrate organs develop from epithelial and mesenchymal tissues, and during their early development they share common morphological features. These include condensation of the mesenchymal cells and thickening, folding or branching of epithelial sheets. Sequential and reciprocal interactions between the epithelial and mesenchymal tissues play central roles in regulation of the morphogenesis of all organs. During recent years increasing amounts of molecular data have accumulated from studies describing developmental changes in expression patterns of molecules, as well as from functional in vitro studies and from the generation of transgenic mice. In this review article, we discuss common features in the molecular regulation that appear to be shared by the developing tooth and other organs. Several growth factors have been shown to act as inductive signals mediating epithelial-mesenchymal interactions in different organs. The early signals are proposed to regulate the expression of master regulatory genes, such as transcription factors. In early tooth germ, bone morphogenetic proteins BMP-2 and BMP-4 regulate expression of the homeobox containing genes Msx-1 and Msx-2. These may specify early patterning of organs through regulation of molecules at the cell surface and the extracellular matrix, such as syndecan-1 and tenascin. Changes in cell adhesion and matrix remodelling, particularly in the organ-specific mesenchyme and in basement membrane contribute to formation of mesenchymal cell condensations and to epithelial morphogenesis. Several growth factors and their receptors, particularly in the TGF beta-, FGF- and EGF- families, have been implicated in formation of mesenchymal condensates and in epithelial morphogenesis of many organs, including the tooth. It is apparent that molecules which regulate morphogenesis in different organs are potential candidate genes for congenital malformation syndromes in which several organs are affected.