Fertilized Mouse Ova Research Articles

223 Lesch-Nyhan syndrome: from patient to mouse model

Abstract Introduction. HPRT1 is a Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) coding gene, mutations of which cause, among other diseases, Lesch-Nyhan syndrome. LNS is a severe X-linked recessive neurological disorder. Alignment shows 98.6% homology of human and murine protein sequences. Objectives. We assume that murine model of this human disease should be developed. So, our purpose is to create a transgenic mouse model of Lesch-Nyhan syndrome by CRISPR/Cas9 genome editing system. Methods. The BLAST was used to find homologous mutation in murine HPRT1 gene comparing to Human one. X-ray crystallographic structural model of the HPRT1 was used as template for M4T Raptor algorithm to generate predicted structure of mutated protein. Models were visualized in PyMol (Schrodinger, Portland, OR). Lack of enzymatic activity of the HGPRT could be caused via troubled homodimerization. Absence of the aliphatic Valine could be the reason of the hindered monomers interaction. Genetic construction based on the px330 plasmid was brought using microinjection method in mouse fertilized ovum for producing primary transgenic organisms. Results. The CRISPR/Cas9 system was specifically designed to carry out mutations in HPRT. The resulting genetic construct was introduced into the fertilized mouse ovum to obtain primary transgenic organisms. This mouse was obtained at the Institute of Biology of the Russian Academy of Sciences. Conclusion. Homologous mutation in human and murine HPRT1 gene resulting into comparable conformational change in the protein model structure was revealed, so murine personalized model of the Lesch-Nyhan syndrome could be developed. Structural changes can be further studied to provide treatment strategy for people suffering from Lesch-Nyhan syndrome. This study was supported by Russian Science Foundation (Grant #17-75-20249).

Biological characteristics of cleft palate relevant gene thyroid transcription factor-2 transgenic mice

The aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered. The C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development. TTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry. The recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.

Amino acid carryover in the subzonal space of mouse fertilized ova affects subsequent transport kinetics

SummaryWe have investigated whether culture in glycine-containing medium affects subsequent glycine transport by the specific transport system, GLYT1, which is the sole glycine transporter in fertilized mouse ova. When fertilized ova were maintained for 6 h in culture with a physiological level of glycine (1 mM), subsequent transport of radiolabelled glycine was decreased by 40% compared with fertilized ova that had been maintained in glycine-free medium. Kinetic measurements showed that the apparent glycine affinity was decreased after culture with glycine (Km increased from 0.20 to 0.41 mM), but maximal transport rate was unchanged (similar Vmax of 20 and 23 fmol/fertilized ovum/min). These findings could have reflected activation of GLYT1 by prolonged substrate starvation, similar to some other amino acid transport systems. However, our findings were instead consistent with the alteration in glycine transport being due to trapping of glycine within the zona pellucida resulting in competitive transport inhibition even after ova were removed from glycine-containing media. First, even very brief exposures to glycine resulted in decreased subsequent glycine transport rates, with a maximal effect apparent within ~6 min. Second, extensive washing (at least six) reversed the effect. Third, the effect was absent when zona-free fertilized ova were used. Thus, it appears that components of the external environment of preimplantation embryos may continue to affect transport kinetics for a period even after embryos are removed from environments that contain them.

Characterization of in vivo recombination activities in the mouse embryo

Homologous recombination makes use of sequence homology to repair DNA and to rearrange genetic material. In mammals, these processes have mainly been characterized using cultured cell systems. We have developed an assay that allows us to quantitatively analyze homologous recombination in vivo in the mouse embryo. Transgenic mouse lines were generated by microinjection into a fertilized mouse ovum of a vector containing two homologous LINE-1 (L1) sequences arranged as a direct repeat: these sequences can recombine with each other and with endogenous L1 sequences before, during or after integration of the vector into the genome. Using a plasmid rescue procedure, we determined the composition of the integrated vector array in several transgenic mice and their descendants. Homologous recombination frequencies were found to be strikingly high, involving 70% of integrated vectors in some arrays, with homologous deletions being five times more frequent than gene conversion without crossing-over. Interestingly, non-homologous recombination was found to be much less frequent. We also found that endogenous L1 sequences could be involved in homologous recombination events in the mouse embryo, and that the integrated arrays could be modified from generation to generation by homologous recombination between the integrated L1 sequences.

Tissue-specific expression of the human neuropeptide Y gene in transgenic mice

Neuropeptide Y (NPY) is the most abundant neuropeptide detected in the mammalian brain, and is found throughout the central and peripheral nervous systems. This peptide is a proposed regulator of appetite, blood pressure, and pituitary hormone release. Previous experiments have demonstrated the ability of 5′ sequences within the human NPY gene to promote transcription in cultured neuronal cells. To identify sequences in this gene that regulate tissue-specific expression, a NPY/CAT fusion gene, containing approximately 850 bp of NPY sequences, was microinjected into fertilized mouse ova. Five lines of transgenic mice were derived from these ova and several tissues from mice of each line were tested for transgene expression using the CAT assay. One line demonstrated X-chromosome-linked transmission of the transgene while the other lines demonstrated autosomally-linked transmission. Three lines demonstrated transgene expression with significant levels of CAT activity detectable only in tissues which have been shown to express endogenous NPY. One autosomally-linked line did not demonstrate significant levels of transgene activity because the transgene appeared to have undergone structural alteration during genomic integration. No transgene activity was detected in either male of female mice from the X-linked line, suggesting a positional regulation of the transgene locus other than X-inactivation in this line. The present research demonstrated the NPY regulatory sequences included i in pCATNPYΔ796 sufficiently directed tissue-appropriate gene expression in transgenic mice.

Developmentally regulated and erythroid-specific expression of the human embryonic β-globin gene in transgenic mice

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.

Introduction to recombinant DNA.

This paper describes the current state of knowledge of methods for analysing gene structure and localization. Illustrations are given of the preparation of complementary DNA libraries and their screening by positive-negative selection, the use of synthetic oligodeoxynucleotides and the use of antibodies. Analysis of the EGF precursor is used as an example to show its close relationship to plasma membrane receptor and its homology with the LDL receptor. Analysis of cloned genome DNA by use of bacteriophage lambda or cosmids gives useful information about gene regulation and evolution. Mutations by frame shift, point or missence mutations are discussed with reference to the LDL receptor and the apolipoproteins. The techniques of gene mapping by rat-human cell hybridization and hybridization in situ are illustrated, again with reference to genes coding for enzymes of cholesterol metabolism, the apolipoproteins and insulin-like growth factors. Finally the potential of in vitro mutagenesis and the injection of cloned DNA into the fertilized mouse ovum are discussed.

Maternal blood platelet physiology and luteal-phase endocrinology as a means of monitoring pre- and postimplantation embryo viability following in vitro fertilization.

The discovery that the fertilized mouse ovum triggers an increased demand for platelets and results in thrombocytopenia during the preimplantation phase of pregnancy provides a monitor for embryo survival and viability. This paper reports a study in which the platelet count was significantly reduced throughout the human preimplantation phase of pregnancy and returned to normal following embryo implantation. The human embryo was shown to produce a platelet activating factor in vitro which caused the reduction in platelet count after embryo transfer. This factor in the embryo culture medium could be measured using a bioassay which provided a means of assessing embryo viability prior to transfer. Some women showed no reduction in platelets after transfer. These embryos failed to produce a platelet activating factor in vitro and pregnancy was not established. Other women displayed a reduction in platelets following transfer but failed to become pregnant. All of these women had elevated luteal-phase plasma E2 levels compared to pregnant patients, which may have interfered with the implantation process. Our observations provide a possible rapid and simple means for monitoring the viability of human embryos cultured in vitro and the survival of embryos in utero.

Rabbit α-globin messenger RNA translation by the mouse ovum

ABSTRACT Fertilized mouse ova were injected with messenger RNA for rabbit globin. The ova were labelled with [3H]leucine and the synthesized rabbit α-globin measured by immunoprecipitation. Injection of rabbit globin mRNA from 4-0 to 15-8pg/ovum resulted in the synthesis of ∼1200dpm/ovum of a-globin during an 18 h incubation. A concentration of mRNA that was translated below the maximum level of globin synthesis was used to determine translation efficiency. The efficiency of translation was 14·1 molecules of a-globin synthesized/cell/h for each molecule of α-globin mRNA injected. The radioactivity in a-globin was 1 % of the total incorporation in acid-precipitable material. Total incorporation of control and injected ova was not significantly different. This study shows that the fertilized mouse ovum can translate an injected foreign message with essentially the same efficiency as that reported for the Xenopus oocyte but substantially lower than the reticulocyte and that the translational capacity of the mouse fertilized ovum for injected mRNA is limited with little if any spare translational ability.

Changes in radiosensitivity of the in vitro fertilized mouse ova during zygotic stage from fertilization to first cleavage.

The radiosensitivity of mouse zygotes fertilized in vitro (BC3F1 ovum x ICR sperm) to X-rays has been measured as a function of time from fertilization to first cleavage. The dose of X-rays (LD50) required to prevent development of 50% of the zygotes to the blastocyst stage in vitro varied markedly depending on the time of irradiation from about 40 to 400 R. Sensitivity is relatively low shortly after sperm entry and becomes extremely high (LD50, 40 R) at the stage just before pronuclear formation (4 to 6 hr after insemination). In the later pronuclear stage (12 hr of fertilization) the sensitivity becomes low (LD50, about 400 R), and it increases again just before first cleavage.

Secretion of proteins by the fertilized mouse ovum

Fertilized one-cell mouse ova were injected with messenger RNA (mRNA) for chicken ovalbumin or rat albumin. The ova were labeled with [ 3H]amino acids, and endogenous proteins as well as proteins in the culture medium were separated by two-dimensional electrophoresis. Following injection of either message, ova were able to synthesize and secrete the appropriate protein. However, when the message for globin was injected, globin appeared only among the endogenous ovum proteins but was not secreted into the culture medium. The location of the secreted proteins, albumin and ovalbumin, on two-dimensional gels suggests that ova process the proteins before secretion by removing the amino-terminal signal sequence from rat albumin and glycosylating ovalbumin. Rat albumin may be exported into the culture medium more rapidly than ovalbumin. In addition, at least one endogenous ovum protein was secreted at the one-cell stage. The studies establish the ability of the newly fertilized mouse ovum to synthesize and secrete proteins from injected as well as endogenous messages.

Surface Alterations of the Mouse Zona Pellucida and Ovum following in vivo Fertilization: Correlation with the Cell Cycle

The zona pellucida and cell surface of in vivo fertilized mouse ova exhibit time dependent changes which can be detected with the scanning electron microscope. The periods of ovulation, fertilization and first cleavage in superovulated C3D2/F/sub 1/ hybrids were determined and times corresponding to G/sub 1/, S, G/sub 2/, and M were calculated. The zona of a mature unfertilized ovum has a rough texture with deep furrows; at fertilization and thereafter the zona develops a smoother, ropy and seemingly porous surface. The cell surface of the unfertilized ovum is characterized by uniform microvilli, small blebs and rounded, mound-like elevations. After fertilization and development to G/sub 1/, the ovum loses its blebs but retains the mound-like elevations and microvilli which are now less uniform. As the ovum progresses toward S, it loses the mound-like elevations but retains microvilli in the same density as found in G/sub 1/. The ovum in G/sub 2/ exhibits smaller but more numerous microvilli which vary considerably in length. Some appear to bifurcate. The fertilized ovum developing through M and G/sub 1/ of the 2 cell stage exhibits a less dense population of relatively uniform microvilli, periodic blebs and, again, rounded elevations. The data are reminiscent ofmore » surface changes associated with the cell cycle in tissue culture cells and indicate a cyclic progression of the in vivo fertilized mouse ovum through the first cleavage division to the 2 cell stage.« less

Response of fertilized mouse ova to freezing and thawing as a function of permeation by glycerol

Response of fertilized mouse ova to freezing and thawing as a function of permeation by glycerol

Infection of mammalian unfertilized and fertilized ova with oncogenic viruses.

REPORTS on the infection of mammalian ova with viruses1–3 chiefly concern the exposure of ova to cytolytic viruses, such as those causing Mengo encephalitis or vesicular stomatitis. We have investigated the effect of two oncogenic viruses, which do not usually destroy infected somatic mouse cells, on unfertilized and fertilized mouse ova.

Studio cinematografico sulle prime fasi di segmentazione dell'uovo di topo

Summary The behaviour of fertilized mouse ova in vitro was observed by means of lime-lapse cinematography: cleavage was followed from the two-cell stage to the formation of the blastocyst. Some types of movements are described, such as those presented by whole blastomeres, as well as the endocytoplasmic movements of different kinds of inclusion. The formation of the blastocyst and its characteristic pulsations are finally described.

Recovery and Culture of Tubal Mouse Ova

FERTILIZED mouse ova have been cultivated in vitro, and their development filmed, by Friedrich-Freksa and Kuhl1, who used as medium a clot of guinea pig plasma and mouse embryo extract containing segments of Fallopian tube2. Like Chang3, I have been working on ovum culture with a view to transplantation of ova. Chang has used rabbits, with serum as a culture medium ; I have chosen to use mice, as more readily available, and because (like most domestic animals) they have naked eggs. Seeking a medium readily prepared in large quantities, I have tried saline hen-egg extracts.