Classical genetics and gene targeting

1999 ◽  
Author(s):  
Scott Bultman ◽  
Terry Magnuson
Keyword(s):  
Gene Targeting ◽  
Cell Lines ◽  
Es Cells ◽  
Coat Colour ◽  
Es Cell ◽  
Allelic Series ◽  
Classical Genetic ◽  
Mutant Phenotypes ◽  
Genomic Regions ◽  
Insight Into

Gene targeting has provided considerable insight into the functions of numerous genes since it was developed a decade ago (1-4). A listing of the diversity of targeted genes and breadth of mutant phenotypes characterized to date can be obtained through mouse mutation databases (http://www.bis.med. jhmi.edu/Dan/tbase.html and http://biomednet.com/cgi-bin/ mko/mkohome.pl) (5, 6). However, in order to take full advantage of this technology, classical genetic methods should be utilized to extend our knowledge of individual genes to genetic pathways. In this chapter, the significance of genetics in gene targeting and phenotype interpretation are discussed. We describe how Mendelian and quantitative genetics can be exploited to map modifier loci or generate animals carrying mutations in two or more genes. We also discuss the development and application of classical genetic approaches towards elucidating gene function such as generation of allelic series and creation of deletion complexes throughout the genome in ES cells and mice. Several genetic considerations should be taken into account during the initial stages of a gene targeting experiment. In order to maximize homologous recombination efficiency, both arms of a targeting vector should be isolated from the same strain of mice as that of the ES cells (see Chapter 1 for details). Although most ES cell lines have been isolated from 129 strains of mice, significant genetic variation exists among the different substrains, which is sometimes evident by pronounced differences in coat colour (see Chapter 4, Table 1) (7, 8). For instance, 129/Sv mice (Aw/Aw, +c-Tyr +p/+c-Tyr +p ) have a white-bellied agouti (Aw) phenotype, whereas 129/SvJ mice (Aw/Aw, Tyrc p/T yrc-ch p) have a cream colour owing to the effect of mutant tyrosinase (Tyr) and pink-eyed dilution (p) alleles which are epistatic to Aw (9). Molecular analysis of different 129 substrains using microsatellite markers has provided insight into their genetic differences and revealed that 129/SvJ is particularly divergent and actually contaminated with genomic regions of non-129 origin (7, 8). Before the significance of this heterogeneity was appreciated, many targeting vectors were constructed from 129/SvJ DNA for use in 129/Sv ES cell lines.

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

scholarly journals Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

2006 ◽  
Vol 17 (11) ◽  
pp. 4675-4685 ◽  
Author(s):  
Linda Yang ◽  
Lei Wang ◽  
Yi Zheng
Keyword(s):  
Gene Targeting ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Cell Model ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Membrane Ruffling ◽  
Primary Mouse

Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian Cdc42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian Cdc42, we have characterized the Cdc42−/− and Cdc42GAP−/− primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the Cdc42 loss- or gain-of-activity cell model. The Cdc42−/− cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the Cdc42GAP−/− cells displayed spontaneous filopodia. The Cdc42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3β, myosin light chain, and FAK phosphorylation. Furthermore, Cdc42−/− cells were defective in G1/S-phase transition and survival, correlating with deficient NF-κB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of Cdc42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that Cdc42 plays cell-type–specific signaling roles.

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.

287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES

2008 ◽  
Vol 20 (1) ◽  
pp. 223 ◽  
Author(s):  
T. Lonergan ◽  
A. Harvey ◽  
J. Zhao ◽  
B. Bavister ◽  
C. Brenner
Keyword(s):  
Cell Lines ◽  
Complex I ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Fibroblast ◽  
Es Cell ◽  
Cell Content ◽  
Stem Cell Lines

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.

Chromatin Profiling of the Globin Loci of Human ES Cells and ES-Derived Erythroid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2535-2535
Author(s):  
Xiangdong Fang ◽  
Kai-Hsin Chang ◽  
Daniel Bates ◽  
Morgan Diegel ◽  
Richard Sandstrom ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Es Cells ◽  
Globin Gene ◽  
Dnase I ◽  
Erythroid Cells ◽  
Es Cell ◽  
Hypersensitive Site ◽  
Human Es Cells ◽  
Chromatin Profiling

Abstract Abstract 2535 Poster Board II-512 We used a high throughput approach to determine the chromatin profiles of the human β and α globin loci and their upstream and downstream regions in human undifferentiated ES cells, ES cell-derived erythroid cells, human fetal and adult origin erythroid cells and in primary cells and cell lines of endo-meso and ectodermal origins. All DNase I hypersensitive sites of the b-locus were absent in undifferentiated human ES cells except for HS2 of the b-globin locus control region. The chromatin profiles of the β and α globin loci of ES cell-derived erythroid cells were identical to those of fetal liver erythroid cells except that the hypersensitive site of the embryonic globin gene was more prominent. DNase I hypersensitive site 2 of the b-globin LCR, a potent enhancer, was present in all the cell lines and primary lineages we studied, providing direct evidence that it is ubiquitous. Several new erythroid specific DHSs were detected upstream of 5′HS7 of the β-LCR, raising the possibility that they play a role in the regulation of the β globin locus. The region downstream to 3′HS1 was depleted of DHSs except for the previously identified DHS mapping near the breakpoint of HPFH 1. Since DHSs are absent near the breakpoints of deletional HPFHs and db thalassemias and since enhancers are typically DHS positive, our results argue against the hypothesis of imported enhancers in the pathogenesis of deletional HPFH and db thalassemia mutants. All the previously identified erythroid specific DHSs of the α globin locus were absent in human ES cells. The α globin locus of ES cells, however, displayed three very prominent DHSs, which were located almost symmetrically about 40 Kb apart from each other and they were constitutively formed in all the lineages and cell lines we have studied; the 3′ and 5′ DHSs carried CTCF sites by ChIP-Seq assay raising the possibility that they mark the sites of chromatin insulators. Overall these results demonstrate the power of the new high throughput chromatin profiling approaches and their ability to uncover features of chromatin that may be of regulatory relevance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Rapid and Highly Efficient Genotyping Pipeline for Screening Gene Targeted Mouse Embryonic Stem Cells

2021 ◽  
Author(s):  
Roger Caothien ◽  
Charles Yu ◽  
Lucinda Tam ◽  
Robert Newman ◽  
Brian Nakao ◽  
...  
Keyword(s):  
Animal Models ◽  
Gene Targeting ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Rapid Screening ◽  
Es Cell ◽  
Screening Process ◽  
Targeting Vector

Abstract Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. The addition of magnetic activated cell sorting (MACS) technology and multiplex droplet digital PCR (ddPCR) to the ES cell screening process can achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. This improved workflow reduces the time needed to generate preclinical animal models. Faster access to animal models for therapeutic target identification and experimental validation can accelerate the development of therapies for human disease.

Efficient Non-Viral Transfection of Mouse and Human Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5267-5267
Author(s):  
Zwi N. Berneman ◽  
Jeremy P. Brown ◽  
Sjaak Van der Sar ◽  
Dave Van den Plas ◽  
Lena Van den Eeden ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Reporter Genes ◽  
Cell Phenotype ◽  
Es Cell ◽  
Short Term ◽  
Human Es Cells ◽  
Mrna Electroporation ◽  
Mouse Es Cell

Abstract BACKGROUND: Development of efficient non-viral gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study we investigated mRNA electroporation as a tool for short-term gene transfer in both mouse and human ES cells. METHODS: Culture and mRNA electroporation conditions for feeder-free cultured mouse and human ES cells were optimized on three mouse ES cell lines (E14, R1 and HM-1) and one human ES cell line (H9). After electroporation with EGFP mRNA, transfected ES cell populations were analyzed by FACS for EGFP expression, viability and phenotype. Also, stably-transfected mouse ES cell lines containing Lox-P or FRT-flanked reporter genes were electroporated with mRNA encoding Cre- or FLPe-recombinase proteins. Monitoring recombination efficiency was done based on the appearance and/or disappearance of fluorescent reporter genes, as determined by FACS analysis. ES cells that underwent recombination were further analyzed for potential to differentiate towards the neural lineage and differentiated cells were analyzed by FACS for expression of neural markers. RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cells resulted in high level transgene expression (>90% EGFP positive cells) combined with low electroporation-induced cell mortality (>90% viable cells). Moreover, the electroporation procedure did not influence ES cell phenotype and further cell culture of undifferentiated ES cell populations. Electroporation of mRNA encoding Cre- or FLPe-recombinase proteins in stably-transfected mouse ES cell lines containing LoxP- or FRT-flanked reporter genes resulted in a recombination efficiency of respectively 75% and 90%. Moreover, these recombination events did not have influence on ES cell phenotype, viability, growth potential, and their ability to differentiate towards neural cell types upon retinoic acid stimulation. (B) Although human ES cells are much more sensitive as compared to mouse ES cells, we were able to develop improved culture and electroporation conditions for feeder-free maintained H9 human ES cells, which resulted in high level transgene expression (>90% EGFP+ cells) combined with high cell viability (>90% viable cells) after EGFP mRNA electroporation. CONCLUSIONS: RNA electroporation is a highly efficient method for short-term genetic loading of both mouse and human ES cells. Ongoing research now focuses on either short-term (via direct mRNA electroporation) or sustained (via mRNA-based FLPe-recombination) expression of transcription factors in ES cells and their influence on cell-fate within in vitro cultured embryoid bodies.

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.

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