Fertilized Mouse Eggs Research Articles

The Upstream Sequence of a New Growth/Differentiation Factor, Midkine (MK), Mediates Developmentally Regulated lac Z Gene Expression in Transgenic Mice.

Midkine (MK) is the product of a retinoic acid responsive gene, and is a heparin binding protein involved in the regulation of growth and differentiation. The 1.9 kb upstream region of MK gene was fused with the bacterial β-galactosidase gene (lac Z) and injected into fertilized mouse eggs. The resulting transgenic mice were used to evaluate the in vivo transcriptional regulation through of the upstream region. Comparison of the β-galactosidase expression and endogenous MK expression indicated that the temporal regulation of the transgene was similar to that of MK gene expression during mouse development. The transgene was neither expressed in the preimplantation period nor in 6.5-day embryos. Transgene expression was high and widely distributed on the 8.5th day, became restricted on the 10.5th and 12.5th days, and thereafter almost confined to the kidney. Thus, the 1.9 kb upstream region accounts for overall temporal regulation of MK gene expression, while there are some differences between the spatial regulation of the transgene expression and that of the endogenous MK gene expression. The transgene was expressed in a few limited regions of the brain of 17 day old embryos, and those sites consisted largely of matrix cells with columnar arrangements. These results suggests a role of MK in the brain development, and MK activity may be involved in retinoic acid induced malformations of the central nervous system.

Regulation of amelogenin gene expression during tooth development

The amelogenins are the predominant matrix proteins in developing enamel and are crucial for proper enamel mineralization. Transgenic mice were constructed in order to identify the segment of the amelogenin gene required for specific expression in enamel organ cells. A 3.5 kb fragment of the bovine X-chromosomal amelogenin gene that includes a TATA box, the transcription initiation site, and 32 bp of exon 1 was linked to the beta galactosidase gene and injected into fertilized mouse eggs. Newborn transgene positive mice expressed beta galactosidase activity in developing teeth treated with the chromogenic substrate Xgal. Foci of ameloblasts were positive in newborn mice; stain intensity and number of positive ameloblasts increased in 1-day and 2-day postnatal mice. Some of the adjacent stratum intermedium cells also were positive in the later stages. Targeting of the transgene to the enamel organ was specific; the only other cells observed to be positive were macrophages, which have endogenous beta galactosidase activity.

The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element.

Production of transgenic mice from in vitro fertilized eggs cryopreserved by ultrarapid freezing

In vitro fertilized mouse eggs (C57BL/6N), followed by ultrarapid freezing were used for production of transgenic mice by microinjection of the chicken beta-actin promoter-driven the firefly luciferase cDNA (beta act-Luc). Following micromanipulation, the survival rates of the cryopreserved eggs and of the fresh in vitro fertilized eggs (control) were 70.8% (131/185) and 71.9% (159/221), respectively. After transferring them into oviducts of psudopregnant recipients on Day 1, 13.6% (17/125) of the cryopreserved eggs developed to live offspring and 14.1% (21/149) of fresh eggs did so. It was confirmed by Southern blotting analysis that each two transgenic mice were produced from the cryopreserved eggs (12%, 2/17) and the fresh eggs (10%, 2/21). All of transgenic mice produced from both eggs showed the expression of the luciferase gene. These results indicate that the in vitro fertilized eggs cryopreserved by ultrarapid freezing, can be, easily and conveniently, used for generation of transgenic mice.

Production of transgenic mice

Transgenic mice are very useful for analyzing the functions of a gene either at the tissue level or at the whole body level. Here we describe the production of transgenic mice by the microinjection of DNA into the pronuclei of fertilized mouse eggs.

Effect of magnetic field on human sperm and fertilized mouse eggs

Effect of magnetic field on human sperm and fertilized mouse eggs

Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a).

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.

Transgenic mouse lines with ectopic expression of alpha-1,3-galactosyltransferase: production and characteristics.

The cDNA of murine alpha-1,3-galactosyltransferase was placed under the control of the beta-actin promoter and cytomegalovirus enhancer, then introduced into male pronuclei of fertilized mouse eggs. The three transgenic mouse lines obtained were analysed for the expression of the transferase by staining with Griffonia simplicifolia agglutinin I-B4 (GSI-B4), which is alpha-galactosyl specific. Compared with wild-type mice, all lines of transgenic mice expressed GSI-B4 binding sites more intensely in the renal tubular brush border and lung alveolar epithelium, and newly expressed them in the photoreceptor outer segments, goblet cells of the small intestine and around spermatogonia. GSI-B4 binding sites were also detected in the liver of some transgenic mice. Even though the introduced enzyme gene was expressed in embryos, it did not severely hinder embryogenesis. The transgenic mice tended to secrete more proteins in the urine than the wild type. Furthermore, low body weights, partial damage to hair growth and early death occurred more frequently in the transgenic mice.

Widespread Distribution of Cells Containing Human DNA in Embryos Derived from Mouse Eggs Injected with Human Chromosome Fragments

The possibility that metaphase chromosomes can serve as a source of genetic material for making transgenic mice was suggested by our previous finding of the incorporation of human satellite DNA into mouse embryos that were injected with microdissected human centromeric fragments. In the present study, we further examined whether this chromosome transfer method can be used to generate transgenic mice containing a portion of human chromosome 4 spanning the Huntington's disease (HD) gene. For this purpose, we used an improved method of metaphase chromosome preparation that may minimize the potential for DNA damage. Using metaphase chromosomes prepared in this manner, chromosome fragments spanning the region of chromosome 4 containing the HD gene were microdissected, retrieved, and injected into fertilized mouse eggs. The injected eggs exhibited good viability and developed with a high efficiency when implanted into foster mothers. To determine whether the human DNA from the injected chromosome fragment had been incorporated into the mouse genome, embryos were harvested at 12.5 days of gestation (dg) and analyzed by in situ hybridization using a human Alu repetitive DNA probe. This analysis showed that most of the embryos contained cells with human Alu repeats. However, all of the embryos were mosaic, and the level of mosaicism was such that we were not able to determine the precise chromosomal origin of the human DNA insert. We discuss the possible basis for the mosaicism and the potential value of such mosaic animals for studying Huntington's disease.

Specific atrial overexpression of G protein coupled human 1 adrenoceptors in transgenic mice

The aim was to develop a transgenic mouse model of atrial beta 1 adrenoceptor overexpression in order to create atrial alteration of the receptor transduction system. Transgenic founders were generated after microinjection of the transgene construct into the pronucleus of fertilised mouse eggs. Heterozygous progeny were screened for RNA expression of the human beta 1 adrenoceptor gene under the control of a 0.56 kb proximal promoter of the human atrial natriuretic factor. One line, out of the three obtained, was selected and further characterised for overexpression of the human beta 1 adrenoceptor. Polymerase chain reaction was employed to detect beta 1 adrenoceptor mRNA, and 125I-cyanopindolol (ICYP) binding assays were used to quantify receptors in heart membranes. A quantitative autoradiographic ICYP binding technique was also used to visualise atrial and ventricular beta adrenoceptors in heart sections. The human beta 1 adrenoceptor was overexpressed specifically in the atria of transgenic mice. The level of the beta 1 adrenoceptor was 5-10-fold higher in transgenic mice compared to basal murine beta 1 adrenoceptors in non-transgenic control mice. Left and right atrial receptor overexpression was confirmed by in vitro autoradiography. The human receptors were able to couple to the murine stimulatory G proteins (Gs), as shown by high affinity binding site dosage using the beta adrenoceptor agonist isoprenaline. Isoprenaline displacement studies allowed the determination of two different affinity sites, one of high affinity (KH = 5.8 nM), and one of low affinity (KL = 520 nM). When expressed in terms of protein density (fmol.mg-1), atrial transgenic beta 1 adrenoceptors displayed a threefold increase in high affinity sites (KH) as compared to control mice. Preliminary electrocardiographic data showed supraventricular premature beats in 6/14 transgenic mice v 2/16 control mice. These transgenic mice may provide a useful pharmacological tool to investigate the pathophysiological consequences of the overactivation of atrial beta 1 adrenoceptor-adenylyl cyclase signalling system.

High-level and erythroid-specific expression of human glucose-6-phosphate dehydrogenase in transgenic mice

Human Glc-6-P dehydrogenase (Glc-6-P) cDNA spanning the entire coding region was subcloned into a pSG5 vector that contains an early SV40 promoter, intron II of the rabbit beta-globin gene, and a polyadenylation signal. This expression cassette was then placed downstream of the human beta-globin locus control region and injected into fertilized mouse eggs. Among five transgenic founders that contained intact copies of the construct, one founder expressed human Glc-6-P dehydrogenase enzyme in a high-level and erythroid-specific fashion (5 x higher than endogenous Glc-6-P dehydrogenase activity). When this male founder mated with a normal individual, all the offspring that carried the transgene showed high-level expression of Glc-6-P dehydrogenase activity in erythroid cells. The endogenous mouse Glc-6-P dehydrogenase in all high-expression mice could be competed out by forming a hybrid with human Glc-6-P dehydrogenase. Our results indicate that the locus control region can drive the human Glc-6-P dehydrogenase gene to be specifically expressed in the erythroid cells of transgenic mice. The results described here provide a basis for experiments designed to express human Glc-6-P dehydrogenase in transgenic mice and suggest a suitable approach to producing a mouse model for studying human Glc-6-P dehydrogenase deficiency.

Okadaic acid induces premature chromosome condensation reflecting the cell cycle progression in one-cell stage mouse embryos.

Haploid parthenogenetic embryos as well as fertilized mouse eggs were treated in vitro with 1-10 microM okadaic acid (OA) at the one-cell stage. Cytogenetic analysis detected that OA induces nuclear envelope breakdown (NEBD) and premature condensation of interphase chromosomes in pronuclei as well as in 2nd polar body (PB) nuclei. G1-, S-, and G2-type prematurely condensed chromosomes (PCC) were found in pronuclei of embryos of different age, which reflects their progression through the first cell cycle. In nuclei from 2nd PBs only G1- and S-type PCC were observed. Using the types of PCC as a criterion of different phases of the cell cycle, it was possible to estimate that in haploid parthenogenetic embryos G1-phase lasts until 5.5 hr post activation (hpa), S-phase takes from 4.5 to 9.5 hpa, and from 8.5 hpa G2-phase had started. Second PBs were found to be in G1-phase until 6.5 hpa and S-phase started in some as early as 5.5 hpa, but in most not before 7.5 hpa. Treatment with OA visualizes G1-chromosomes in pronuclei as well as in 2nd PBs, and it is easy to count the number of these chromosomes and recognize a T6 marker chromosome. The possibility to apply cytogenetic analysis of G1-chromosomes from 2nd PBs for a more accurate detection of maternal meiotic nondisjunction is discussed.

Transient expression of a human β‐actin promoter/lacZ gene introduced into mouse embryos correlates with a low degree of methylation

We have analysed the correlation between expression and methylation for the human beta-actin promoter introduced into mouse embryos. The beta-actin promoter was fused to the reporter gene lacZ, and expression was analysed after pronuclear injection into fertilized mouse eggs. We analysed transient expression in in vitro cultured preimplantation embryos and expression after chromosomal integration in 5 independent lines of transgenic mice. The in vitro cultured preimplantation embryos expressed lacZ from the 2-cell to the blastocyst stages, and most abundantly at the morula stage. By increasing the amount of injected DNA, a larger proportion of embryos expressed lacZ. Embryos expressing lacZ in only a subset of the blastomeres were detected at all preimplantation stages. In contrast to the transient expression after injection, we have not detected lacZ expression in any of the 5 analysed lines of transgenic mice carrying the same construct. The lack of expression in transgenic mice correlates with hypermethylation of C residues in the vast majority of CG sequences in the integrated beta-actin/lacZ construct, whereas the injected construct was completely nonmethylated. We discuss methylation and other possible reasons for the observed differences in expression between injected and integrated copies of the beta-actin/lacZ construct and for lacZ expression in only a subset of blastomeres in preimplantation embryos.

The majority of G0 transgenic mice are derived from mosaic embryos

Most transgenic mice are generated by the direct microinjection of DNA fragments into the pronuclei of fertilized eggs. It has been generally assumed that the majority of integration events occur prior to the first round of chromosomal DNA replication (Palmiter and Brinster, 1986). In this study we have determined by comparison of PCR, Southern blot and transmission frequencies that at least 62% of integration events generate a mosaic (somatic and/or germline) G0 transgenic mouse. Furthermore, the statistical probability of transgene-containing cells segregating to the various early embryo lineages implies that this is probably an underestimate of the true mosaic frequency. Thus, the majority of DNA injected into fertilized mouse eggs intergates after the first round of chromosomal DNA replication, therefore most G0 transgenic mice are derived from a mosaic embryo.

Oncogene expression in mammary epithelial cells.

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer-promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter-oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of individual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.

Immunocytochemical localization of cyclin/proliferating cell nuclear antigen (PCNA) in fertilized eggs of mice.

Immunolocalization of cyclin/PCNA (proliferating cell nuclear antigen) was performed with monoclonal antibody using immunogold methods on ultrathin cryosections of fertilized mouse eggs. Immunolabeling in pronuclei was checked 20, 22, 24 and 26 h after HCG injection. A relation between onset of pronuclei migration (early S-phase) and appearance of colloidal particle clusters was found. Afterwards, (mid S-phase) the increase of labelling and the localization of cyclin/PCNA were found throughout the pronuclei, except in the nucleolar bodies. Lower labelling appeared at the time of close reciprocal pronuclei contact (late S-phase). It is concluded that bulk and distribution of cyclin/PCNA in pronuclei is closely related to the progression of first interphase after fertilization.

Expression of SV40‐lacZ gene in mouse preimplantation embryos after pronuclear microinjection

In order to study the expression of an exogenous gene in developing mouse embryos during the preimplantation period, DNA carrying the SV40 early promoter fused with the Escherichia coli beta-galactosidase gene (lacZ) was microinjected into the pronucleus of fertilized mouse eggs. Expression of lacZ gene was detected by staining embryos with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a substrate at pH 7.2. The embryos expressing the lacZ gene showed various intensities of blue staining, all showing a mosaic pattern. The exogenous gene was expressed from the 4-cell stage until the blastocyst stage. The proportion of embryos expressing the lacZ gene was maximal (38%) at the morula stage, and the expression was dependent on the presence of the SV40 promoter.

Reconstruction of a large DNa fragment from coninjected small fragments by homologous recombination in fertilized mouse eggs

Reconstruction of a large DNa fragment from coninjected small fragments by homologous recombination in fertilized mouse eggs

Recognition of the CDEl motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development.

In vitro survival of cells derived from isodiploid uniparental half embryos of the mouse after aggregation with normal embryos

Fertilized mouse eggs, heterozygous for two allozymes of glucose phosphate isomerase (GPI) were bisected, and the resulting half eggs were diploidized with cytochalasin B. After separate aggregation with normal embryos carrying a third allozyme of GPI, the resulting chimaeras were kept in culture up to 10 days. The majority grew out on the culture dish during this period. By GPI analysis, 7.7% of the embryos were found to be chimaeric. Both types of uniparental cells, from gynogenetic and from androgenetic half eggs, were capable of surviving in chimaeras in vitro. These results are comparable with published data obtained by using uniparental embryos generated by micromanipulation.