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.

290 A CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINE DERIVED FROM A SINGLE BLASTOMERE

2008 ◽  
Vol 20 (1) ◽  
pp. 224
Author(s):  
J. Okahara-Narita ◽  
J. Yamasaki ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
K. Wakimoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cynomolgus Monkey ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Cell Stage ◽  
Vitro Assay ◽  
Single Blastomere ◽  
Cell Colony

The establishment of most embryonic stem (ES) cell lines requires the destruction of embryos. Some ES cell lines in mice and humans are currently derived from a single blastomere, so that remaining blastomeres can still develop into fetuses. However, the procedures currently in use for establishing these lines are very complicated, and other ES cell lines from the same species are needed (Chung et al. 2006 Nature 439, 216–219; Klimanskaya et al. 2006 Nature 444, 481–485). The objective of this study was to devise a method simpler than those previously described for establishing ES cell lines from a single blastomere in the cynomolgus monkey. Controlled ovarian stimulation and oocyte recovery have been described previously by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI), and then cultured at 38�C in 5% CO2, 5% O2 for 2 days. The zona pellucida of 4- to 5-cell-stage embryos was disrupted using acidic Tyrode's solution, and individual blastomeres were separated from the denuded embryos using trypsin. These blastomeres were cultured on mitomycin-C-treated mouse embryonic fibroblasts and ES medium containing adrenocorticotropic hormone (ACTH) (Ogawa et al. 2004 Genes to Cells 9, 471–477). After the formation of initial outgrowths, half of the medium was changed every other day until the outgrowths reached approximately 100 cells. Passage of putative monkey ES cells was performed by mechanical dispersion of the colonies and transfer to fresh feeders every 3–4 days until there were enough cells for enzymatic dispersion. One stable ES cell line was obtained from two 4- or 5-cell-stage embryos using ES medium containing ACTH. The morphology of this ES cell colony was consistent with the monkey ES cell colony previous described by Suemori et al. (2001 Dev. Dynamics 222, 273–279). The ES cell line was passaged more than 17 times, and the morphology of the ES cell colony did not differ between the first and seventeenth passages. The ES cells showed normal karyotype and retained pluripotency markers for primate ES cells including octamer-binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, and TRA-1-81. We are presently confirming whether this ES cell line possesses potencies to differentiate in all three embryonic germ layers using both an in vitro assay and teratoma formation. Here we showed that cynomolgus monkey ES cells can be derived from a single blastomere, without co-culturing another ES cell line, as has been done in previous studies on mice and humans. This method allows the establishment of ES cell lines from a single blastomere, leaving the other blastomeres available for embryo transfer. Thus, the method described here is simpler than previously described methods and alleviates some ethical concerns.

scholarly journals Characterization of a novel embryonic stem cell line from an ICSI-derived blastocyst in the African green monkey

Reproduction ◽  
2010 ◽  
Vol 139 (3) ◽  
pp. 565-573 ◽  
Author(s):  
Nobuhiro Shimozawa ◽  
Shinichiro Nakamura ◽  
Ichiro Takahashi ◽  
Masanori Hatori ◽  
Tadashi Sankai
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Biomedical Research ◽  
Es Cells ◽  
Cell Monolayer ◽  
Embryonic Stem ◽  
African Green Monkey ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Green Monkey

Several cell types from the African green monkey (Cercopithecus aethiops), such as red blood cells, primary culture cells from kidney, and the Vero cell line, are valuable sources for biomedical research and testing. Embryonic stem (ES) cells that are established from blastocysts have pluripotency to differentiate into these and other types of cells. We examined an in vitro culture system of zygotes produced by ICSI in African green monkeys and attempted to establish ES cells. Culturing with and without a mouse embryonic fibroblast (MEF) cell monolayer resulted in the development of ICSI-derived zygotes to the blastocyst stage, while culturing with a buffalo rat liver cell monolayer yielded no development (3/14, 21.4% and 6/31, 19.4% vs 0/23, 0% respectively; P<0.05). One of the nine blastocysts, which had been one of the zygotes co-cultured with MEF cells, formed flat colonies consisting of cells with large nuclei, similar to other primate ES cell lines. The African green monkey ES (AgMES) cells expressed pluripotency markers, formed teratomas consisting of three embryonic germ layer tissues, and had a normal chromosome number. Furthermore, expression of the germ cell markers CD9 and DPPA3 (STELLA) was detected in the embryoid bodies, suggesting that AgMES cells might have the potential ability to differentiate into germ cells. The results suggested that MEF cells greatly affected the quality of the inner cell mass of the blastocysts. In addition, AgMES cells would be a precious resource for biomedical research such as other primate ES cell lines.

scholarly journals Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

1983 ◽  
Vol 3 (11) ◽  
pp. 2076-2088 ◽  
Author(s):  
F Ardeshir ◽  
E Giulotto ◽  
J Zieg ◽  
O Brison ◽  
W S Liao ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Syrian Hamster ◽  
Mutant Cell ◽  
Screening Method ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Gene Coding ◽  
Cad Gene ◽  
Mutant Cells

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate

1983 ◽  
Vol 3 (11) ◽  
pp. 2076-2088
Author(s):  
F Ardeshir ◽  
E Giulotto ◽  
J Zieg ◽  
O Brison ◽  
W S Liao ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Syrian Hamster ◽  
Mutant Cell ◽  
Screening Method ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Gene Coding ◽  
Cad Gene ◽  
Mutant Cells

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.

A novel retrovirally induced embryonic lethal mutation in the mouse: assessment of the developmental fate of embryonic stem cells homozygous for the 413.d proviral integration

Development ◽  
1991 ◽  
Vol 111 (4) ◽  
pp. 969-981 ◽  
Author(s):  
F.L. Conlon ◽  
K.S. Barth ◽  
E.J. Robertson
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Lethal Mutation ◽  
Es Cell ◽  
Wild Type ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Embryonic Lethal ◽  
Mutant Cells

A genetic screen of transgenic mouse strains, carrying multiple copies of an MPSV neo retroviral vector, has led to the identification of a recessive embryonic lethal mutation, termed 413.d. This mutation is associated with a single proviral insertion and when homozygous, results in the failure of the early postimplantation embryo at the gastrulation stage of development. Embryonic stem cell lines (ES cells) were derived from 413.d intercross embryos. Genotyping, with respect to the 413.d integration site, identified wild-type, heterozygous and homozygous ES cell lines. The differentiation abilities and developmental potential of the ES cell lines were assessed using a number of in vitro and in vivo assays. Results indicate that the ES cell lines, regardless of genotype, are pluripotent and can give rise to tissue and cell types derived from all three germ layers. Furthermore, analysis of midgestation conceptuses (10.5 p.c.) and adult chimeras generated by injecting mutant ES cells into host blastocysts, provides strong evidence that the mutant cells can contribute to all extraembryonic tissues and somatic tissues, as well as to functional germ cells. These results indicate that the homozygous mutant cells can be effectively ‘rescued’ by the presence of wild-type cells in a carrier embryo.

Oct-4 Is Crucial for Murine Embryonic Stem Cell Secretion of Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis of Murine ES Cells and Bone Marrow Hematopoietic Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1359-1359
Author(s):  
Ying Guo ◽  
Barbara Graham-Evans ◽  
Charlie R. Mantal ◽  
Robert A. Hromas ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Caspase 3 ◽  
Es Cells ◽  
Embryonic Stem ◽  
Conditional Knockout ◽  
Es Cell ◽  
Wild Type ◽  
Murine Bone Marrow

Abstract Murine embryonic stem (ES) cells may be of potential use for cell replacement and gene therapy. Maintenance of ES cells in an undifferentiated and proliferative state depends on cytokines either secreted by ES cells and/or added to the medium. By understanding the production and release of cytokines in ES cell culture, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that SDF-1/CXCL12, secreted by ES cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells (Guo et al, Stem cells, in press, 2005). To evaluate whether other cytokines were produced by murine ES cells, we generated conditioned medium (CM) from these cells in the presence of LIF, while the ES cells were in an undifferentiated Oct-4 expressing state, and assayed the CM for cytokines, chemokines, and other growth modulatory factors. ES cell CM enhanced survival in vitro of ES cells subjected to delayed addition of serum to ES cell cultures. Without serum, ES cells didn’t grow in low cell density. However, with CM, ES cells formed colonies at about 63% of the growth of the ES cells in the presence of serum. ES cell CM also enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased the rate of apoptosis in murine bone marrow c-kit+lin− cells as assessed by Annexin V assay. Our data showed ES cell CM contained IL-1α, IL-10, IL-11, M-CSF, OSM, SCF, VEGF, as well as a number of chemokines and other proteins. For a number of these proteins, we have already verified that the mRNA for them is expressed in the ES cells. This indicates that ES cells produce and secrete these cytokines. Some of these cytokines are known to have an enhanced survival/antiapoptosis effect on progenitors. IL-6, FGF-9, and TNF-a, which were not detected prior to irradiation of the ES cells, were seen after ES cells were irradiated. Irradiation of the ES cells enhanced release of some proteins and decreased release of others. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for cytokines released from ES cells which supported CFU-GM survival and antiapoptosis. Oct-4 conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. CM from the wild type ES cell line enhanced survival of CFU-GM similar to that of other ES cell lines, while the Oct-4 knockout ES cell line didn’t. These results indicate that release of proteins involved in survival enhancement may be related to Oct-4 expression. We also found that the wild type cell line which expressed Oct-4 didn’t initiate caspase 3 dependent apoptosis after mitotic stress. However ZHBTc4, the Oct-4 deleted cell line demonstrated caspase 3 dependent apoptosis. These results may be of physiological significance, although this has not yet been proven, and suggest the possibility of potential future applicability for use of irradiated ES cells as accessory cells for growth modulation in vitro and in vivo.

scholarly journals Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells

2003 ◽  
Vol 369 (3) ◽  
pp. 461-468 ◽  
Author(s):  
Eric C. CARLSON ◽  
Kazuhisa MAMIYA ◽  
Chia-Yang LIU ◽  
Robert L. GENDRON ◽  
David E. BIRK ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Mutant Cell ◽  
Dendritic Morphology ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Stable Transformants ◽  
Collagen Fibrillogenesis

The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys37-Xaa3-Cys41-Xaa-Cys-Xaa9-Cys) and a Cys→Ser (C/S) mutant (Cys37-Xaa3-Ser41-Xaa-Cys-Xaa9-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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

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