Embryonic stem cell-mediated transfer and correct regulation of the chicken delta-crystallin gene in developing mouse embryos

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 259-269 ◽  
Author(s):  
Y. Takahashi ◽  
K. Hanaoka ◽  
M. Hayasaka ◽  
K. Katoh ◽  
Y. Kato ◽  
...  
Keyword(s):  
High Efficiency ◽  
Piriform Cortex ◽  
Embryonic Stem ◽  
Mutant Gene ◽  
Mouse Embryos ◽  
Es Cell ◽  
Wild Type ◽  
Flanking Sequence ◽  
Mouse Tissues ◽  
Type Gene

To study regulation of delta-crystallin expression during ontogeny, we transferred the gene from chicken into developing mouse embryos by first transforming an embryonic stem (ES) cell line of mouse and then producing chimaeric embryos by combining them with normal mouse embryos. Using this technique, genes were transferred into a variety of developing mouse tissues with high efficiency. Two delta-crystallin gene constructs were used: the wild-type gene with 2200 bp of the 5′ flanking sequence, shown to be lens-specific in an assay using cultured mouse cells, and a mutant gene with 51 bp of the 5′ flanking sequence, lacking the sequence required for expression in lens cells. Five independent lines carrying the former and two lines carrying the latter were employed in producing chimaeras. In the chimaeric embryos having the wild-type gene, delta-crystallin was expressed in the lens and in specific regions of the primitive central nervous system (CNS) as is seen in embryonic expression in the chicken. In adult mouse chimaeras also, expression was restricted to the lens and the CNS, in the pyramidal neurones of the piriform cortex and the hippocampus. delta-crystallin expression in these tissues is due to proper transcriptional regulation, since no expression was observed when chimaeras were produced with the ES lines carrying the mutant gene. The experimental results reported here demonstrate the advantage of ES-cell-mediated gene transfer in the study of embryonic gene regulation, because a number of gene constructs and chromosomal sites can be analysed shortly after embryo manipulation without requiring gene transmission to the next generation.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

scholarly journals A Shortened Life Span of EKLF−/− Adult Erythrocytes, Due to a Deficiency of β-Globin Chains, Is Ameliorated by Human γ-Globin Chains

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1291-1299 ◽  
Author(s):  
Sai-Kiang Lim ◽  
James J. Bieker ◽  
Chyuan-Sheng Lin ◽  
Frank Costantini
Keyword(s):  
Life Span ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Es Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Mature Erythrocyte ◽  
Heinz Bodies ◽  
Globin Chains

Abstract Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF−/− ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF−/− colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of βh1-globin, which continued to be expressed at a very low level. The ratio of adult β-globin/α-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF−/− cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF−/− erythrocytes in adult animals must be short-lived, apparently due to the imbalance of β-versus α-globin chains, leading to the precipitation of excess α-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF−/− ES cells of a human LCR/γ-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

scholarly journals Tumor Suppression by a Severely Truncated Species of Retinoblastoma Protein

2002 ◽  
Vol 22 (9) ◽  
pp. 3103-3110 ◽  
Author(s):  
Hong Yang ◽  
Bart O. Williams ◽  
Phillip W. Hinds ◽  
T. Shane Shih ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Tumor Suppression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Retinoblastoma Protein ◽  
Pituitary Tumors ◽  
Es Cell ◽  
Chimeric Mice ◽  
Wild Type ◽  
Substantial Fraction

ABSTRACT Rb+/+:Rb−/− chimeric mice are healthy until early in adulthood when they develop lethal pituitary tumors composed solely of Rb−/− cells. In an effort to delineate the minimal structures of the retinoblastoma protein necessary for RB tumor suppression function, chimeric animals derived from stably transfected RB−/− embryonic stem (ES) cells were generated. One such ES cell transfectant expressed a human RB allele encoding a stable, truncated nuclear derivative lacking residues 1 to 378 (Δ1-378). Others encoded either wild-type human RB or an internally deleted derivative of the Δ1-378 mutant. All gave rise to viable chimeric animals with comparable degrees of chimerism. However, unlike control mice derived, in part, from naive Rb−/− ES cells or from ES cells transformed by the double RB mutant, Δ1-378/Δexon22, animals derived from either wild-type RB- or Δ1-378 RB-producing ES cells failed to develop pituitary tumors. Thus, in this setting, a substantial fraction of the RB sequence is unnecessary for RB-mediated tumor suppression.

Unique Primitive Erythropoiesis Defect In Rpl5-Deficient Murine Embryonic Stem Cell Model of Diamond Blackfan Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 877-877
Author(s):  
Tracie A. Goldberg ◽  
Sharon Singh ◽  
Adrianna Henson ◽  
Abdallah Nihrane ◽  
Jeffrey Michael Lipton ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Es Cells ◽  
Mutant Cell ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Es Cell ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Primitive Erythropoiesis

Abstract Abstract 877 Background: Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods: We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results: We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel). For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion: Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). Disclosures: No relevant conflicts of interest to declare.

Knockdown of p53 Improves Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem Cell Models of Diamond Blackfan Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 728-728 ◽  
Author(s):  
Tracie A. Goldberg ◽  
Sharon Singh ◽  
Jonathan Solaimanzadeh ◽  
Jeffrey Goldstein ◽  
Jeffrey Michael Lipton ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Mutant Cell ◽  
Embryonic Stem ◽  
Es Cell ◽  
Cell Models ◽  
Wild Type ◽  
Cell Stage ◽  
P53 Expression ◽  
Diamond Blackfan Anemia

Abstract Abstract 728 Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. The disease has been shown to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, may play a role in the pathogenesis of DBA. Previously, we described murine embryonic stem (ES) cell models of DBA and reported hematopoietic and erythroid defects common to Rps19- and Rpl5-deficient cell lines, as well as a primitive erythropoiesis defect unique to an Rpl5-deficient cell line [Blood 116(21), 877, 2010]. Methods: We studied the effects of p53 knockdown on hematopoiesis in our Rps19- and Rpl5-mutant murine ES cell lines created by gene trap technology. Small interfering RNA (siRNA) targeting p53 was transfected into mutant cell lines at the ES cell stage. A non-targeting siRNA served as a negative control. After 24 hours, cells were plated into methylcellulose medium with fetal bovine serum and stem cell factor (SCF) to generate embryoid bodies (EBs). On day 7, EBs were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 12 for total quantity and hematopoietic percentage. For secondary differentiation into primitive erythroid colonies, day 5 EBs were disrupted, and individual cells were suspended in a methylcellulose medium containing fetal bovine plasma-derived serum and epo. Primitive erythroid colonies were counted on day 7 of culture. Definitive hematopoiesis assays were performed by disruption of day 7 EBs, followed by suspension of cells in methylcellulose medium containing SCF, IL-3, IL-6 and epo. Definitive hematopoietic colonies were counted on day 10. In an independent set of experiments, we created an isogenic pair of wild-type and mutant DBA ES cells by electroporation of another Rps19- mutant line with a plasmid vector expressing wild-type Rps19 cDNA (wild-type) or an empty vector (mutant). Results: By immunoblot assays, we detected an increased amount of p53 protein in our Rps19-and Rpl5- mutant cell lines. Following p53 siRNA transfection, we confirmed 82–95% reduction in p53 expression by quantitative PCR, whereas ES cells transfected with non-targeting siRNA did not alter p53 expression. For both Rps19- and Rpl5- mutants, previously shown to have EB formation defects in comparison to parental controls, p53 knockdown significantly improved EB formation, especially hematopoietic-type EBs, compared to mutants treated with non-targeting siRNA. In addition, p53 knockdown in both mutants reversed the definitive hematopoiesis defect by increasing the ratio of erythroid colony to myeloid colony formation. Furthermore, p53 siRNA transfection of the Rpl5- mutant rescued the primitive erythropoiesis defect previously shown by us. To further explore the mechanistic basis of our findings, we additionally tested the effects of Rpl11 knockdown in our DBA models. The presence of free RPL11 secondary to abortive ribosome assembly has been hypothesized to be responsible for increased p53 in DBA by binding to and inhibiting the p53 inhibitor HDM2 (Mdm2 in mice). Transfection of Rpl11 siRNA into both Rps19- and Rpl5-mutant cell lines at the ES cell stage led to a marked reduction in EB formation, compared to cells transfected with non-targeting siRNA. Finally, we also extended our analysis to an isogenic pair of Rps19- wild-type and mutant cells. In the mutant line, we confirmed a 5–8 fold rescue of EB formation with siRNA targeting p53 when compared to the non-targeting siRNA. In order to clarify the role of two major downstream effectors of p53, siRNA targeting either Bax or p21 was transfected into the mutant cell line. Surprisingly, neither siRNA was able to rescue the EB formation defect of the mutant cells. Conclusions: (1) Knockdown of p53 markedly improves erythroid defects of Rps19- and Rpl5-deficient murine ES cell models of DBA, while inhibition of the upstream target Rpl11 causes significant toxicity to cells already haploinsufficient for Rps19 or Rpl5. (2) Knockdown of either Bax or p21 does not recapitulate knockdown of p53, suggesting that neither plays a significant individual role in downstream signaling from p53 in this model. (3) Further exploration of the p53 pathway may provide insights into the pathogenesis of DBA and identify new targets for therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibited Neurogenesis in JNK1-Deficient Embryonic Stem Cells

2005 ◽  
Vol 25 (24) ◽  
pp. 10791-10802 ◽  
Author(s):  
Claudia R. Amura ◽  
Lindsay Marek ◽  
Robert A. Winn ◽  
Lynn E. Heasley
Keyword(s):  
Pc12 Cell ◽  
Neural Differentiation ◽  
Es Cells ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Signal Pathways ◽  
Es Cell ◽  
Wild Type ◽  
Base Pairs ◽  
Promoter Sequences

ABSTRACT The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.

182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 190
Author(s):  
T.-Y. Fu ◽  
P.-C. Tang
Keyword(s):  
Expression Pattern ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Cell Stage ◽  
Morula Stage

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 653-660 ◽  
Author(s):  
D. Huszar ◽  
A. Sharpe ◽  
S. Hashmi ◽  
B. Bouchard ◽  
A. Houghton ◽  
...  
Keyword(s):  
Neural Crest ◽  
Retroviral Vector ◽  
Mouse Embryos ◽  
Pigment Cells ◽  
Wild Type ◽  
Tyrosinase Gene ◽  
History Of ◽  
Albino Mice ◽  
Type Gene ◽  
Dual Origin

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the ‘standard’ stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.

scholarly journals Immunocytochemical Analyses and Targeted Gene Disruption of GTPBP1

2000 ◽  
Vol 20 (17) ◽  
pp. 6195-6200 ◽  
Author(s):  
Satoru Senju ◽  
Ken-ichi Iyama ◽  
Hironori Kudo ◽  
Shinichi Aizawa ◽  
Yasuharu Nishimura
Keyword(s):  
Smooth Muscle ◽  
Gene Disruption ◽  
Amino Acid Level ◽  
Embryonic Stem ◽  
Mutant Mice ◽  
Wild Type ◽  
Targeted Gene Disruption ◽  
Mouse Tissues ◽  
Significant Difference ◽  
Deficient Mice

ABSTRACT We previously identified a gene encoding a putative GTPase, GTPBP1, which is structurally related to elongation factor 1α, a key component of protein biosynthesis machinery. The primary structure of GTPBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1. Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that GTPBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages. Immunofluorescence analyses revealed that GTPBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages. To investigate the physiological role ofGTPBP1, we used targeted gene disruption in embryonic stem cells to generate GTPBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in GTPBP1-deficient mice may be due to functional compensation by GTPBP2, a molecule we recently identified which is similar to GTPBP1 in structure and tissue distribution.

scholarly journals Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1121-1133 ◽  
Author(s):  
Helen Priddle ◽  
Lance Hemmings ◽  
Susan Monkley ◽  
Alison Woods ◽  
Bipin Patel ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Es Cell ◽  
Wild Type

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

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