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Autor     Luciana M. Fontanari Krause
Titel    Identification and functional characterization of the human and murine OSTL gene, which encodes a RING-DRIL-RING domain protein possibly involved in B cell differentiation and leukemogenesis
Ort    München
Jahr    2006
Anmerkung    Date Accepted: 29. May 2006, Deposited On:19. Jun 2006, Note: this source as well as the thesis come with the date "2006". Nevertheless it seems likely that this source is in fact the source of the copying: see Vpr/Befunde/Fontanari_Krause
URL    http://edoc.ub.uni-muenchen.de/5368

Literaturverz.   

no
Fußnoten    no
Fragmente    17


Fragmente der Quelle:
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1.5 Chromosomal Translocations

Recurring chromosomal abnormalities have been identified in a variety of cancers, but are most frequently associated with leukemias, lymphomas and sarcomas (Rabbitts, 1994; Rowley, 1999). At present, more than 500 recurring [cytogenetic abnormalities have been reported in hematological malignancies.]


Rabbitts, T. H. (1994). Chromosomal translocations in human cancer. Nature 372, 143-149.

Rowley, J. D. (1999). The role of chromosome translocations in leukemogenesis. Semin Hematol 36, 59-72.

4.2. Chromosomal translocations

Recurring chromosomal abnormalities have been identified in a variety of cancers, but are most frequently associated with leukemias, lymphomas and sarcomas (Rabbitts, 1994; Rowley, 1999). At present, more than 500 recurring cytogenetic abnormalities have been reported in hematological malignancies.


Rabbttis T. H. (1994). Chromosomal translocations in human cancer. Nature. 372 (6502), 143-9.

Rowley J. D. (1999). The role of chromosome translocations in leukemogenesis. Semin Hematol. 4 (7), 59-72.

Anmerkungen

Text is identical, the source is not mentioned.

The copied text is continued on the next page: Vpr/Fragment_016_01

Sichter
(Hindemith) Schumann

[2.] Vpr/Fragment 016 01 - Diskussion
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[At present, more than 500 recurring] cytogenetic abnormalities have been reported in hematological malignancies. Three main cytogenetic changes have been detected in hematological malignancies: deletions, inversions and translocations. At present, more than 500 recurring cytogenetic abnormalities have been reported in hematological malignancies. Three main cytogenetic changes have been detected in hematological malignancies: deletions, inversions and translocations.
Anmerkungen

The text is identical, the source is not mentioned.

The copied text starts on the previous page: Vpr/Fragment_015_26.

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(Hindemith) Schumann

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There are at least seven different chromosomal translocations associated with a myeloproliferative disease. These include the BCR/ABL, ETV6/ABL, ETV6/PDGFRB, HIP1/PDGFRB, H4/PDGFRB, RAB5E/PDGFRB, and ETV6/JAK2 fusions (table 1) (Dash and Gilliland, 2001).

Table 1: Translocations involving tyrosine kinases

MPD = myeloproliferative disease

16a diss Vpr.png


Dash, A., Gilliland, D. G. (2001) Molecular genetics of acute myeloid leukaemia. Best Pract Res Clin Haematol 14, 49-64.

There are at least seven different chromosomal translocations associated with myeloproliferative disease. These include the BCR/ABL, ETV6/ABL, ETV6/PDGFRB, HIP1/PDGFRB, H4/PDGFRB, RAB5E/PDGFRB, and ETV6/JAK2 fusions (Table 4.1 adapted from Dash, 2001).

Table 4.1: Chromosomal translocations involving tyrosine kinases

16a source Vpr.png

MPD = myeloproliferative disease


Dash A., Gilliland D. G. (2001). Molecular genetics pf [sic] acute myeloid leukaemia. Best Pract Res Clin Haematol. 14, 49-64.

Anmerkungen

The source is not given.

Note that in Dash & Gilliland (2001) the table and the text cannot be found in this form.

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(Hindemith) Schumann

[4.] Vpr/Fragment 019 01 - Diskussion
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The failure of certain fusion genes to induce leukemia on their own in animal models as well as the fact that more than 50% of acute myeloid leukemias do not have apparent cytogenetic abnormalities, point to the role of point mutations in or, alternatively, the aberrant expression of proto-oncogenes in the development of leukemia. These observations also suggest that the aberrant expression of proto-oncogenes might be more common than is generally believed. The sheer number of ETV6 fusions in AML allows for the study of a diverse group of leukemias in which more than one of the aforementioned mechanisms might be operating. The t(12;13) translocation is particularly interesting in this regard as the expression of both the ETV6/CDX2 fusion gene as well as the ectopic expression of the CDX2 gene has been demonstrated.

1.6 ETV6

ETV6 (ETS variant gene 6) was originally called TEL, for translocation ets leukemia gene. ETV6 is a member of the ets (E-26 transforming specific) family of transcription factors. All ets family proteins share a conserved protein domain of about 88 amino acids in length, the so called ets domain (see Figure 10) (Bohlander SK 2005). The ets domain is a sequence specific DNA binding domain but it also mediates protein-protein interaction. The other evolutionarily conserved domain is the N terminally located pointed or SAM (sterile alpha motif) domain in the 652 amino acids of ETV6 (Bohlander SK 2005). This domain is also called HLH (helix loop helix) domain. It is found in yeast proteins and has been shown to be involved in homo and heterodimerization of transcription factors and in signal transducing proteins (e.g. of the MAPK pathway) (Fig.10). ETV6 contains two alternative translational start codons (position 1 and position 43), leading to the expression of two isoforms of ETV6.


Bohlander S K (2005). ETV6: a versatile player in leukemogenesis.Semin Cancer Biol 15(3):162-74.

The failure of certain fusion genes to induce leukemia on their own, in animal models, as well as the fact that more than 50% of acute myeloid leukemias do not have apparent cytogenetic abnormalities, point to the role of point mutations in or, alternatively, the aberrant expression of proto-oncogenes in the development of leukemia. These observations also suggest that the aberrant expression of proto-oncogenes might be more common than is generally believed.

The sheer number of ETV6 fusions in AML allows for the study of a diverse group of leukemias in which more than one of the aforementioned mechanisms might be operating. The t(6;12) translocation is one of several that involve ETV6, in this case ETV6 is fused to STL gene in a B-cell ALL cell line.

4.3. ETV6

ETV6 is a member of the ets (E-26 transforming specific) family of transcription factors. All ets family proteins share a highly conserved protein domain of about 88 amino acids in length the so-called ets domain. The ets domain is a sequence specific DNA binding domain but is [sic] also mediates protein-protein interaction (Figure 4.7, modified from Slupsky, 1998), it is

[page 21]

evolutionarily highly conserved and found in invertebrates such as Drosophila and C. elegans (Oikawa, 2003; Wasylyk, 1993).

The other evolutionarily conserved domain in the 652 amino acids of ETV6 is the N-terminally located pointed or sterile alpha motif (SAM) domain (Figure 4.7). This domain is also called HLH domain and is even more highly conserved in evolution and found in many ets family member. It is found in yeast proteins and has been shown to be involved in homo and heterodimerization in transcription factors and in signal transducing proteins (e.g. of the MAPK pathway) (Grimshaw, 2004). ETV6 contains two alternative translational start codons (position 1 and position 43) leading to the expression of two isoforms of ETV6.

Anmerkungen

The source is not given.

Note that the text of the second part of the fragment can also be found in Bohlander (2005), see Vpr/Dublette/Fragment 019 13. However, despite the fact that Vpr mentions Bohlander (2005) in the text, the likely source is still the here documented Fontanari Krause (2006), because the text there is closer to Bohlander (2005), in particular containing text fragments not included in Vpr (e.g. the half-sentence: "and is even more highly conserved in evolution and found in many ets family member"), such that it is not likely that Fontanari Krause (2006) copied from Vpr, more likely is the other way round. It is also unlikely that both authors copied from Bohlander (2005) independently as there are passages in close proximity that can be found in Vpr and in Fontanari Krause (2006) but not in Bohlander (2005)

Sichter
(SleepyHollow02), (Hindemith), Schumann

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Figure 10. ETV6 protein structure.

1.6.1 Role of ETV6 in hematopoiesis

ETV6 is widely expressed throughout embryonic development and in the adult. Embryos with a conventional knockout (KO) of the Etv6 gene die by day 11 of embryonic development (E11) due to vascular abnormalities. Blood formation in the embryo is largely unperturbed (Wang et. al., 1997). Yet, studies using chimeric mice from Etv6-deficient embryonic stem (ES) cells suggested a requirement of Etv6 in bone marrow hematopoiesis. Inducible and lineagespecific gene disruption of Etv6 in adult hematopoiesis in mice suggests that it plays two important roles in hematopoietic differentiation. First, Etv6 controls the survival of HSCs so that its disruption indirectly affects the majority of all hematopoietic cells which have limited clonal life spans and eventually will extinguish without constant regeneration from HSCs. Secondly, Etv6 is required late in the development of the megakaryocyte lineage, where it presumably acts in concert with transcriptional regulators previously implicated in megakaryopoiesis (Hock et. al., 2004).

1.6.2 ETV6 fusion partners

Translocations involving the ETV6 gene contribute to leukemogenesis through at least 3 different mechanisms. One mechanism is the activation of kinases. The second mechanism is the loss of function of critical transcription factors and/or the formation of aberrant transcription factors and the third mechanism is the induction of ectopic and aberrant expression of the proto-oncogene by the [chromosomal translocation.]


Hock, H., Meade, E., Medeiros, S., Schindler, J. W., Valk, P. J., Fujiwara, Y., and Orkin, S. H. (2004). Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival. Genes Dev.

Wang, L. C., Kuo, F., Fujiwara, Y., Gilliland, D. G., Golub, T. R., and Orkin, S. H. (1997). Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL. Embo J 16, 4374-4383.

[page 21]

20a source Vpr.png

Figure 4.7: ETV6 protein structure. The pointed domain is represented between Nt 38 and Nt 123; ETS domain is between Nt 338 and Nt 422 (modified from Slupsky, 1998).

4.3.1. Role of ETV6 in hematopoiesis

ETV6 is widely expressed throughout embryonic development and in the adult. Embryos with a conventional knockout (KO) of the Etv6 gene die by day 11 of embryonic development (E11) due to vascular abnormalities. Blood formation in the embryo is largely unperturbed (Wang, 1997). Yet, studies using chimeric mice from Etv6-deficient embryonic stem (ES) cells suggested a requirement of Etv6 in bone marrow hematopoiesis. Inducible and lineagespecific gene disruption of Etv6 in adult hematopoiesis in mice suggests that it plays two important roles in hematopoietic differentiation. First, Etv6 controls the survival of HSCs so that its disruption indirectly affects the majority of all hematopoietic cells which have limited clonal life spans and eventually will extinguish without constant regeneration from HSCs.

[page 22]

Secondly, Etv6 is required late in the development of the megakaryocyte lineage, where it presumably acts in concert with transcriptional regulators previously implicated in megakaryopoiesis (Hock, 2004).

4.3.2. ETV6 fusion partners

Translocations involving the ETV6 gene contribute to leukemogenesis through at least 3 different mechanisms. One mechanism is the activation of kinases. The second mechanism is the loss of function of critical transcription factors and/or the formation of aberrant transcription factors and the third mechanism is the induction of ectopic and aberrant expression of the proto-oncogene by the chromosomal translocation.


Hock H., Meade E., Medeiros S., Schindler J. W., Valk P. J., Fujiwara Y., Orkin S. H. (2004). Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival. Genes Dev. 18 (19), 2336-41.

Wang L. C., Kuo F., Fujiwara Y., Gilliland D. G., Golub T. R., Orkin S. H. (1997). Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL. EMBO J. 16, 4374-83.

Anmerkungen

The source is not mentioned. For the figure neither the original source Slupsky (1998) is mentioned.

Note that much of section 1.6.1 can also be found verbatim in Hock et al. (2004). This includes the reference to Wang (1997).

Sichter
(SleepyHollow02) Schumann, Hindemith

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Figure 11. This is a schematic diagram representing the various breakpoints of ETV6 (indicated by arrows) in the translocations involving different partner genes (indicated in closed boxes)

21b source Vpr.png

Figure 4.8: Diagram of ETV6 with protein domain and breakpoints (indicated by arrows) representing different partner genes of ETV6 (indicated in closed boxes) (adapted from Bohlander, 2005).

Anmerkungen

The image is copied from the source without this being made clear.

Note that the same figure can also be found in the original source Quelle:Vpr/Bohlander_2005, see Vpr/Dublette/Fragment_021_03. Inspecting the figure caption one can see that the caption of Fontanari Krause (2006) lies somewhat in between the versions of Vpr and Quelle:Vpr/Bohlander_2005, which suggests that Vpr adapted it from Fontanari Krause (2006) who adapted it from Quelle:Vpr/Bohlander_2005.

The next figure (Figure 12) that also comes from the source is marked with: "(kindly provided by Prof. Stefan Bohlander)".

Sichter
(Hindemith) Schumann

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1.6.3 Protein tyrosine kinase fusion partners of ETV6

The first identified fusion partner of ETV6 was a protein tyrosine kinase (PTK), the platelet-derived growth factors receptor beta (PDGFRB) (Golub et. al., 1994). The fusion protein critical for the development of the chronic myelo-[monocytic leukemia is the ETV6/PDGFRB fusion and not the reciprocal PDGFRB/ETV6 fusion.]


Golub, T. R., Barker, G. F., Lovett, M., and Gilliland, D. G. (1994). Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 77, 307-316.

4.3.3. Protein tyrosine kinase fusion partners of ETV6

The first identified fusion partner of ETV6 was a protein tyrosine kinase (PTK), the platelet-derived growth factors receptor beta (PDGFRB) (Golub, 1994a). The fusion protein critical for the development of the chronic myelomonocytic leukemia is the ETV6/PDGFRB fusion, and not the reciprocal PDGFRB/ETV6 fusion.


Golub, T. R., Barker G.F., Lovett M., Gilliland D. G. (1994a). Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell. 77 (2), 307-16.

Anmerkungen

The source is not mentioned here.

The copied text continues on the next page Vpr/Fragment_022_01.

Note that the text is also quite similar to another source, see: Vpr/Dublette/Fragment_021_10

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(Hindemith) Schumann

[8.] Vpr/Fragment 022 01 - Diskussion
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[The fusion protein critical for the development of the chronic myelo-]monocytic leukemia is the ETV6/PDGFRB fusion and not the reciprocal PDGFRB/ETV6 fusion. In the ETV6/PDGFRB fusion protein the N terminal portion of ETV6, which includes the pointed domain, is fused to the C-terminal two thirds of the PDGFRB protein, conserving the tyrosine kinase domain of PDGFRB. The fusion of the pointed domain of ETV6 in the N-terminal half with the tyrosine kinase domain in the C-terminal half of the fusion partner is characteristic of the class of ETV6/PTK fusions and is found in the fusions of ETV6 with ABL1, ABL2, JAK2, NTRK3, FGFR3 and SYK (Fig. 11) (Table 2) (Papadopoulos et. al., 1995; Cazzaniga et. al., 1999; Knezevich et. al., 1998; Kuno et. al., 2001; Peeters et. al., 1997)

Table2. Tyrosine kinase fusion partners of ETV6

22a diss Vpr.png

1.6.4 Transcription factors and other fusion partners of ETV6

The ETV6/RUNX1 (ETV6/AML1) fusion is the most common fusion gene in childhood acute B cell lymphoblastic leukemia (Shurtleff et. al., 1995).


Cazzaniga, G., Tosi, S., Aloisi, A., Giudici, G., Daniotti, M., Pioltelli, P., Kearney, L., and Biondi, A. (1999). The tyrosine kinase abl-related gene ARG is fused to ETV6 in an AML-M4Eo patient with a t(1;12)(q25;p13): molecular cloning of both reciprocal transcripts. Blood 94, 4370- 4373.

Knezevich, S. R., McFadden, D. E., Tao, W., Lim, J. F., and Sorensen, P. H. (1998). A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat Genet 18, 184-187.

Kuno, Y., Abe, A., Emi, N., Iida, M., Yokozawa, T., Towatari, M., Tanimoto, M., and Saito, H. (2001). Constitutive kinase activation of the TEL-Syk fusion gene in myelodysplastic syndrome with t(9;12)(q22;p12). Blood 97, 1050-1055.

Papadopoulos, P., Ridge, S. A., Boucher, C. A., Stocking, C., and Wiedemann, L. M. (1995). The novel activation of ABL by fusion to an ets-related gene, TEL. Cancer Res 55, 34-38.

Peeters, P., Raynaud, S. D., Cools, J., Wlodarska, I., Grosgeorge, J., Philip, P., Monpoux, F., Van Rompaey, L., Baens, M., Van den Berghe, H., and Marynen, P. (1997a). Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia. Blood 90, 2535- 2540.

Peeters, P., Wlodarska, I., Baens, M., Criel, A., Selleslag, D., Hagemeijer, A., Van den Berghe, H., and Marynen, P. (1997b). Fusion of ETV6 to MDS1/EVI1 as a result of t(3;12)(q26;p13) in myeloproliferative disorders. Cancer Res 57, 564-569.

Shurtleff, S. A., Buijs, A., Behm, F. G., Rubnitz, J. E., Raimondi, S. C., Hancock, M. L., Chan, G. C., Pui, C. H., Grosveld, G., and Downing, J. R. (1995). TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia 9, 1985-1989.

The fusion protein critical for the development of the chronic myelomonocytic leukemia is the ETV6/PDGFRB fusion, and not the reciprocal PDGFRB/ETV6 fusion. In the ETV6/PDGFRB fusion protein the N terminal portion of ETV6, which includes the pointed domain, is fused to the C-terminal two thirds of the PDGFRB protein, conserving the tyrosine kinase domain of PDGFRB. The fusion of the pointed domain of ETV6 in the N-terminal half with the tyrosine kinase domain in the C-terminal half of the fusion partner is characteristic of the class of ETV6/PTK fusions

[page 23]

and is found in the fusions of ETV6 with ABL1, ABL2, JAK2, NTRK3, FGFR3 and SYK (Table 4.2 adapted from Bohlander, 2005) (Papadopoulos, 1995; Cazzaniga, 1999; Kuno, 2001).

Table 4.2: Protein tyrosine kinase fusion partner of ETV6

22b source Vpr.png

4.3.4. Transcription factors and other fusion partners of ETV6

The ETV6/RUNX1 (ETV6/AML1) fusion is the most common fusion gene in childhood acute B cell lymphoblastic leukemia (Shurtleff, 1995).


Bohlander S. K. (2005). ETV6: A versatile player in leukemogenesis. Semin Cancer Biol. 15 (3), 162-74.

Cazzaniga G., Tosi S., Aloisi A., et al. (1999). The tyrosine kinase abl-related gene ARG is fused to EYV6 in an AML-M4Eo patient with a t(1;12)(q25;p13): molecular cloning of both reciprocal transcripts. Blood. 94, 4370-73.

Kuno Y., Abe a., Emi N., Iida M., Yokozawa t., towatari M., et al. (2001). Constitutive kinase activation of the TEL-Syk fusion gene in myelodysplastic syndrome with t(9;12)(q22;p12). Blood. 97, 1050-5.

Papadopoulos P., Rigde S. A., Boucher C. A., Stocking C., Wiedemann L. M. (1995). The novel activation of ABL by fusion to an ets-related gene, TEL. Cancer Res. 55 (1), 34-8.

Shurtleff S. A., Buijs A., Behm F. G., Rubnitz J. E., Raimondi S. C., Hancock M. L., Chan G. C., Pui C. H., Grosveld G., Downing J. R. (1995).TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent. Leukemia. 9 (12), 1985-9.

Anmerkungen

The source is not mentioned here.

Note that the table in the source Fontanari Krause (2006) has the same columns as Vpr but a different order of the columns. The original source Bohlander (2005) has the same column order as Fontanari Krause (2006), but two additional columns that appear neither in Vpr nor in Fontanari Krause (2006). This can be interpreted as indication that Vpr copied from Fontanari Krause (2006) who copied from Bohlander (2005).

Note that there are two publicationen "Peeters et. al., 1997" in the bibliography.

Note also that Vpr is closer to Fontanari Krause (2006) than to Bohlander (2005), see Vpr/Dublette/Fragment_022_01. However, Vpr includes two references: Knezevich et al. (1998) and Peeters et al. (1997) that also Bohlander (2005) mentions, but Fontanari Krause (2006) does not.

Sichter
(Hindemith) Schumann

[9.] Vpr/Fragment 023 01 - Diskussion
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[1.6.4 Transcription factors and other fusion partners of ETV6

The ETV6/RUNX1 (ETV6/AML1) fusion is the most common fusion gene in childhood acute B cell lymphoblastic leukemia (Shurtleff et. al., 1995). Re-]porter gene assays showed that the ETV6/RUNX1 fusion protein acts as a repressor by binding to the promoter and enhancer regions of RUNX1 target genes. This repression function is dependent on the pointed and on the central domain of ETV6, which are both part of the ETV6 portion of the ETV6/RUNX1 fusion protein (Fenrick et. al., 1999). ETV6/ARNT and HLXB9/ETV6 chimeric proteins are other examples of this class of ETV6 fusions found in AML and the HLXB9/ETV6 hybrid is detected in up to 20% of pediatric cases with AML (Beverloo et al., 2001) A potential mechanism of transformation for these fusions is that the ETV6/ARNT and HLXB9/ETV6 proteins interact with the wild-type ETV6 through the pointed domain, thereby interfering with normal ETV6 function (Beverloo et. al., 2001).

1.6.5 Ectopic and aberrant expression of a proto-oncogene gene

A number of ETV6 translocations including the ETV6-MDS1/EVI1 and the ETV6-CDX2 fusion only contain the transcription/translation start of ETV6 (Peeters et al., 1997, Chase et al., 1999). In these cases ectopic expression of the transcription factors EVII and CDX2 was detected in addition to the expression of the fusion gene. The potential importance of the ectopic expression of a proto-oncogene in this class of ETV6 fusions was further underlined by observations that the ectopic expression of the proto-oncogene also occurred when the fusion gene itself was not translated into a protein product. For example patients with the t(4;12) positive leukemia show ectopic expression of the ParaHox gene GSH2. However the fusion gene CHIC2-ETV6 generated by the chromosomal translocation was not translated into a protein product. Furthermore, expression of IL-3 was observed in a CML case with a t(5;12), lacking the ETV6-ACS2 fusion protein. These results suggest that ectopic expression of GSH2 and IL-3 could be the key leukemogenic mechanism in these leukemias (Cools et. al., 2002).


Beverloo, H. B., Panagopoulos, I., Isaksson, M., van Wering, E., van Drunen, E., de Klein, A., Johansson, B., and Slater, R. (2001). Fusion of the homeobox gene HLXB9 and the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13). Cancer Res 61, 5374-5377.

Chase, A., Reiter, A., Burci, L., Cazzaniga, G., Biondi, A., Pickard, J., Roberts, I. A., Goldman, J. M., and Cross, N. C. (1999). Fusion of ETV6 to the caudal-related homeobox gene CDX2 in acute myeloid leukemia with the t(12;13)(p13;q12). Blood 93, 1025-1031.

Cools, J., Mentens, N., Odero, M. D., Peeters, P., Wlodarska, I., Delforge, M., Hagemeijer, A., and Marynen, P. (2002). Evidence for position effects as a variant ETV6-mediated leukemogenic mechanism in myeloid leukemias with a t(4;12)(q11-q12;p13) or t(5;12)(q31;p13). Blood 99, 1776- 1784.

Fenrick, R., Amann, J. M., Lutterbach, B., Wang, L., Westendorf, J. J., Downing, J. R., and Hiebert, S. W. (1999). Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein. Mol Cell Biol 19, 6566-6574.

Peeters, P., Raynaud, S. D., Cools, J., Wlodarska, I., Grosgeorge, J., Philip, P., Monpoux, F., Van Rompaey, L., Baens, M., Van den Berghe, H., and Marynen, P. (1997a). Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia. Blood 90, 2535- 2540.

Peeters, P., Wlodarska, I., Baens, M., Criel, A., Selleslag, D., Hagemeijer, A., Van den Berghe, H., and Marynen, P. (1997b). Fusion of ETV6 to MDS1/EVI1 as a result of t(3;12)(q26;p13) in myeloproliferative disorders. Cancer Res 57, 564-569.

Shurtleff, S. A., Buijs, A., Behm, F. G., Rubnitz, J. E., Raimondi, S. C., Hancock, M. L., Chan, G. C., Pui, C. H., Grosveld, G., and Downing, J. R. (1995). TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia 9, 1985-1989.

[page 23]

4.3.4. Transcription factors and other fusion partners of ETV6

The ETV6/RUNX1 (ETV6/AML1) fusion is the most common fusion gene in childhood acute B cell lymphoblastic leukemia (Shurtleff, 1995). Reporter gene assays showed that the ETV6/RUNX1 fusion protein acts as a repressor by binding to the promoter and enhancer regions of RUNX1 target genes. This repression function is dependent on the pointed and on the central domain of ETV6, which are both part of the ETV6 portion of the ETV6/RUNX1 fusion protein (Fenrick, 1999). ETV6/ARNT and HLXB9/ETV6 chimeric proteins are other examples of this class of ETV6 fusions found in AML and the HLXB9/ETV6 hybrid is detected in up to 20% of pediatric cases with AML (Beverloo, 2001). A potential mechanism of transformation for these fusions is that the ETV6/ARNT and HLXB9/ETV6 proteins interact with the wild-type ETV6 through the pointed domain, thereby interfering with normal ETV6 function (Beverloo, 2001).

4.3.5. Ectopic and aberrant expression of a proto-oncogene gene

A number of ETV6 translocations including the ETV6-MDS1/EVI1 and the ETV6-CDX2 fusion only contain the transcription/translation start of ETV6 (Peeters, 1997; Chase, 1999). In these cases ectopic expression of the transcription factors EVI1 and CDX2 was detected in addition to the expression of the fusion gene.

[page 24]

The potential importance of the ectopic expression of a proto-oncogene in this class of ETV6 fusions was further underlined by observations that the ectopic expression of the proto-oncogene also occurred when the fusion gene itself was not translated into a protein product: an example for this is the ectopic expression of the ParaHox gene GSH2 in patients with t(4;12) positive leukemia, even in cases, in which no protein expression of the CHIC2-ETV6 fusion generated by the chromosomal translocation, could be detected. Furthermore, expression of IL-3 was observed in a CML case with a t(5;12), lacking the ETV6-ACS2 fusion protein. These results suggest that ectopic expression of GSH2 and IL-3 could be the key leukemogenic mechanism in these leukemias (Cools, 2002).


Beverloo H. B., Panagopoulos I., Isaksson M., et al. (2001). Fusion of the homeobox gene HLXB9 and the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13). Cancer Res. 61, 5374-77.

Chase A., Reiter A., Burci L., et al. (1999). Fusion of ETV6 to the caudal-related homeobox gene CDX2 in acute myeloid leukemia with the t(12;13)(p12;q12). Blood. 93, 1025-31.

Cools J., Mentens N., Odero M. D., et al. (2002). Evidence for position effects as a variant ETV6- mediated leukemogenic mechanism in myeloid leukemias with a t(4;12)(q11-q12;p13) or t(5;12)(q31;p13). Blood. 99, 1776-84.

Fenrick R., Amann J. M., Lutterbach B., et al. (1999). Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein. Mol Cell biol. 19, 6566-74.

Peeters P., Raynaud S. D., Cools J., et al. (1997). Fusion of TEL. the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a mueloid leukemia. Blood. 90, 2535-40.

Shurtleff S. A., Buijs A., Behm F. G., Rubnitz J. E., Raimondi S. C., Hancock M. L., Chan G. C., Pui C. H., Grosveld G., Downing J. R. (1995).TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent. Leukemia. 9 (12), 1985-9.

Anmerkungen

The source is not mentioned.

Note that the first two sentences can also be found in Bohlander (2005), but Fontanari Krause (2006) is the more likely source.

Note that there are two references "Peeters et al., 1997" in the bibliography.

Sichter
(SleepyHollow02) Schumann, Hindemith

[10.] Vpr/Fragment 030 13 - Diskussion
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For retroviral gene transfer into primary bone marrow (BM) cells, the different constructs were subcloned into the multiple cloning site of the modified MSCV 2.1 vector (Pineault et. al., 2003b) upstream of the internal ribosomal entry site (IRES) and the enhanced green or yellow fluorescent protein (GFP/YFP) gene. As a control the MSCV vector carrying only the IRESEGFP cassette was used.

Pineault, N., Buske, C., Feuring-Buske, M., Abramovich, C., Rosten, P., Hogge, D. E., Aplan, P. D., and Humphries, R. K. (2003). Induction of acute myeloid leukemia in mice by the human leukemia- specific fusion gene NUP98-HOXD13 in concert with Meis1. Blood 101, 4529-4538.

For retroviral gene transfer into primary bone marrow (BM) cells, the murine OSTL (clone J16) was subcloned into the multiple cloning site of the modified MSCV 2.1 vector (Pineault et al., 2003) upstream of the internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (GFP) gene (2.2.7.1). As a control, the empty MSCV vector carrying was used.

Pineault N., Buske C., Feuring-Buske M., Abramovich C., Rosten P., Hogge D. E., Aplan P. D., Humphries R. K. (2003). Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1. Blood. 101 (11), 4529-38.

Anmerkungen

The source is not mentioned.

Note that there is no "Pineault et. al., 2003b" in the list of references.

Sichter
(Hindemith) Schumann

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3.3 Retrovirus Production.

Retrovirus was produced with the constructs mentioned before by individually co-transfecting 15 μg of the construct DNA with equal amounts of Ecopac DNA into 293T cells by using the calcium chloride precipitation method of transfection (Pineault et. al., 2003b). Virus conditioned medium (VCM) was collected 48h after transfection at time intervals of eight hours for forty-eight hours. Production of high-titer helper-free retrovirus was achieved by transducing the ecotropic packaging cell line GP+E86 (Pineault et al., 2003b) with virus conditioned medium (VCM) from 293T transfected cells. Successfully transduced GP+E86 cells were sorted out by FACS using GFP or YFP as a marker 4 days after transduction using standard procedures (Pineault et. al., 2003a).


Pineault, N., Buske, C., Feuring-Buske, M., Abramovich, C., Rosten, P., Hogge, D. E., Aplan, P. D., and Humphries, R. K. (2003). Induction of acute myeloid leukemia in mice by the human leukemia- specific fusion gene NUP98-HOXD13 in concert with Meis1. Blood 101, 4529-4538.

[page 90]

5.10.2. Retrovirus Production

Retrovirus was produced with the constructs mentioned above by co-transfecting 30 μg of the construct plasmid DNA with equal amounts of Ecopac DNA into 293T cells using the

[page 91]

calcium phosphate precipitation method (Pineault et al., 2003). Virus conditioned medium (VCM) was collected 48h after transfection at time intervals of eight hours for forty-eight hours. Production of high-titer helper-free retrovirus was achieved by transducing the ecotropic packaging cell line GP+E86 (Pineault et al., 2003) with virus conditioned medium (VCM) from 293T transfected cells. Successfully transduced GP+E86 cells were sorted out by FACS using GFP as a marker 4 days after transduction using standard procedures (Pineault et al., 2003).


Pineault N., Buske C., Feuring-Buske M., Abramovich C., Rosten P., Hogge D. E., Aplan P. D., Humphries R. K. (2003). Induction of acute myeloid leukemia in mice by the human leukemiaspecific fusion gene NUP98-HOXD13 in concert with Meis1. Blood. 101 (11), 4529-38.

Anmerkungen

The source is not given.

Note that there is only one source "Pineault et al. (2003) listed in the bibliography.

Note that in Pineault et al. (2003) (the paper listed in the bibliography) the here documented parallel text cannot be found.

Sichter
(Hindemith) Schumann

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3.4 Retroviral infection of primary BM cells.

Primary mouse bone marrow (BM) cells were transduced as previously described (Pineault et. al., 2003a). Briefly, BM cells were obtained by flushing both femurs and tibias of donor mice treated 4 days previously with 150 mg/kg 5-fluorouracil injected into the tail vein. 5-flourouracil eliminates cycling cells from the animal, thus enriching the bone marrow for primitive hematopoietic progenitors, which are non-cycling or quiescent in nature (Reya et. al., 2001). Cells were stimulated for 48 hrs in DMEM supplemented with 15% FBS, 10 ng/ml mIL-6, 6 ng/ml mIL-3 and 100 ng/ml murine stem cell factor (SCF). For transduction, cells were co-cultured for 48h with irradiated (40 Gy) GP+E86 virus producing cells in the same medium with an addition of 5 μg/ml protamine sulfate with ETV6/CDX2/GFP or Cdx2/YFP GP+E86 producers or with a mixture of 40 to 50% CDX2/YFP and 50 to 60% ETV6/CDX2/GFP producers in co-transduction experiments. Protamine sulfate prevents aggregation of viral particles, thus increasing efficiency of transduction. Loosely adherent and nonadherent BM cells were harvested from the co-culture 48h post transduction, BM cells were furtherer cultured in fresh medium with cytokine cocktail for 48h to allow expression of EGFP or EYFP. Transduced BM cells were sorted out by florescence activated cell sorting (FACS) using EGFP or EYFP as a marker.

In Vitro Assays

3.5 Proliferation Assay.

To study the proliferative potential in vitro of BM cells transduced with ETV6/CDX2, Cdx2, its mutants and ETV6/CDX2 + Cdx2, we performed a proliferation assay by plating an equal number of successfully transduced bone marrow cells directly after sorting in DMEM supplemented with 15% FBS, 10 ng/ml mIL-6, 6 ng/ml mIL-3 and 100 ng/ml mSCF (standard medium) (Tebubio GmbH, Offenbach, Germany) at 370C in a humidified CO2 incubator. The [cells were subjected to half-media change every 7 days and their proliferation assessed on the same day by counting viable cells after trypan exclusion.]

[page 91]

5.10.3. Retroviral infection of primary BM cells

Primary mouse bone marrow (BM) cells were transduced as previously described (Pineault et al., 2003). Briefly, BM cells were obtained by flushing both femurs and tibias of donor mice treated 4 days previously with 150 mg/kg 5-fluorouracil (myeloid condition) injected into the tail vein. Mice without 5-fluorouracil treatment (for lymphoid condition) were also used in this work. 5-flourouracil eliminates cycling cells hematopoietic cells from the animal, thus enriching the bone marrow for primitive hematopoietic progenitors, which are non-cycling or quiescent in nature (Reya, 2001). Cells were stimulated for 48 hrs in DMEM supplemented with 15% FBS, 10 ng/ml IL6, 6 ng/ml IL3 and 10 ng/ml mSCF.

For lymphoid condition assays, cells were stimulated for 24 h in DMEM supplemented with 15% FBS, 10 ng/ml IL7, 6 ng/ml mutant FLT3 and 10 ng/ml mSCF.

For transduction, cells were co-cultured for 48h with irradiated (40 Gy) GP+E86 virus producing cells in the same medium with the addition of 5 μg/ml protamine sulfate. Protamine sulfate prevents aggregation of viral particles, thus increasing efficiency of transduction. Loosely adherent and non-adherent BM cells were harvested from the coculture 48h post transduction, BM cells were furtherer cultured in fresh medium with cytokine cocktail for 48 h or 24 h (myeloid or lymphoid condition) to allow expression of EGFP. Transduced BM cells were sorted by fluorescence activated cell sorting (FACS) using EGFP as a marker.

5.10.4. In Vitro Assays

5.10.4.1. Proliferation Assay

To study the proliferative potential in vitro of BM cells transduced with OSTL expressing retroviruses, a proliferation assay was performed by plating successfully transduced bone marrow cells directly after sorting in DMEM supplemented with 15% FBS, 10 ng/ml mIL-

[page 92]

6, 6 ng/ml mIL-3 and 100 ng/ml murine stem cell factor (SCF) (standard medium) (Tebubio GmbH, Offenbach, Germany) at 37°C in a humidified 5% CO2 incubator. The cells were subjected to half-media change every 7 days and their proliferation assessed on the day of media change by counting viable cells after trypan exclusion.

Anmerkungen

The source is not mentioned here.

Sichter
(SleepyHollow02) Schumann

[13.] Vpr/Fragment 034 01 - Diskussion
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[The] cells were subjected to half-media change every 7 days and their proliferation assessed on the same day by counting viable cells after trypan exclusion. To generate IL-3 dependent cell lines, successfully transduced bone marrow cells were cultured directly after sorting in DMEM, 15% FBS with mIL-3 alone (6ng/ml). A half-media change was done every 7 days.

3.6 Colony Forming Cells Assay (CFC-assay).

To check the differentiation and clonogenic potential of the BM cells expressing the various genes mentioned above, we performed a CFC assay by testing colony formation in methylcellulose. The CFC–assay was performed by culturing highly purified transduced cells (500 /dish) in 35mm-diameter Petri dishes directly after sorting in 1ml methylcellulose supplemented with cytokines (Methocult M3434), and incubated at 370C in humidified CO2 incubator. Colonies were counted microscopically on 7 to 9 days after plating according to standard criteria (Schwaller et. al., 1998). Re-plating capacity of clonogenic progenitors was assayed by re-plating the primary colonies on secondary methylcellulose dishes. Again colonies were counted day at 7 to 9 after the secondary re-plating.

The cells were subjected to half-media change every 7 days and their proliferation assessed on the day of media change by counting viable cells after trypan exclusion. To generate IL-3 dependent cell lines, successfully transduced bone marrow cells were cultured directly after sorting in DMEM, 15% FBS with IL-3 alone (6 ng/ml). A half-media change was done every 7 days.

5.10.4.2. Colony Forming Cells Assay (CFC-assay)

To analyze the differentiation and clonogenic potential of the transduced BM cells, we performed CFC assays by testing colony formation in methylcellulose. The CFC–assay was done by culture highly purified transduced cells (500/dish) in 35mm-diameter Petri dishes directly after sorting in 1ml methylcellulose supplemented with cytokines (Methocult M3434). Colonies were counted microscopically on 7 to 9 days after plating according to standard criteria (Schwaller, 1998). Re-plating capacity of clonogenic progenitors was assayed by replating the primary colonies (500 cells/dish) on secondary methylcellulose dishes. Again colonies were counted at 7 to 9 days after the secondary re-plating.

Anmerkungen

The source is not mentioned here.

Sichter
(SleepyHollow02) Schumann

[14.] Vpr/Fragment 035 01 - Diskussion
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3.8 Cyto-Morphology.

Cyto-morphological analysis of bone marrow was done by transferring 5x104 to 1x105 bone marrow cells on glass slide by centrifugation at 500 rpm for 10 minute using a Shandon Cytospin2 centrifuge. Slides were stained with May- Grunwald’s eosine-methylene blue and Giemsa solution using the standard protocol supplied by Merck.

3.9 Immunophenotyping:

Cell differentiation was determined and lineage distribution was checked by immunophenotyping. 5x104 to 1x104 cells were rinsed with PBS and stained for 30 minutes on ice with an appropriate concentration of phycoerythrin (PE) conjugated antibody to Gr-1, Sca1, Ter-119, CD4, and allophycocyanin-conjugated antibody for lineage markers Mac-1, cKit, B220 or CD8. The cells were then washed with PBS and stained with propidium iodide, and viable cells were analyzed with the FACS Calibur system.

[page 92]

5.10.5. Cyto-Morphology

Cyto-morphological analysis of bone marrow cells was done by transferring 5x104 to 1x105 bone marrow cells on glass slide by centrifugation at 500 rpm for 10 minute using a Shandon Cytospin2 centrifuge. Slides were stained with May-Grunwald’s eosine-methylene blue and Giemsa solution using the standard protocol supplied by Merck.

5.10.6. Immunophenotyping

Cell differentiation was determined and lineage distribution was analyzed by immunophenotyping. 5x104 to 1x104 cells were rinsed with PBS and stained for 30 minutes on ice with an appropriate concentration of phycoerythrin (PE) conjugated antibody to Gr- 1, Sca-1, Ter-119, CD4, and allophycocyanin-conjugated antibody for lineage markers Mac-1, cKit, B220 or CD8 (all Pharmingen Heidelberg, Germany). The cells were then

[page 93]

washed with PBS and stained with propidium iodide, and viable cells were analyzed with the FACS Calibur system.

Anmerkungen

The source is not given.

Sichter
(Hindemith) Schumann

[15.] Vpr/Fragment 036 07 - Diskussion
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3.12 BM transplantation and assessment of mice.

For bone marrow transplantation procedures, 8–10 week old recipient F1 (B6C3) mice were irradiated with 850 cGy from a 137Cs [sic] γ-radiation source. FACS purified transduced BM cells or defined ratios of transduced and untransduced cells were injected into the tail vein of irradiated recipient mice. PB or BM cell progeny of transduced cells were tracked using GFP or YFP fluorescence (Feuring-Buske et al., 2002). Lineage distribution was determined by flow cytometric analysis as previously described (Pineault et. al., 2003): Phycoerythrin-( PE) labeled Gr-1, Sca1, Ter-119, CD4 and allophycocyanin (APC) labeled Mac1, c-Kit, B220, CD8 antibodies were used for analysis (all Pharmingen Heidelberg, Germany). For histological analyses, sections of selected organs were prepared and H&E stained at the Academic Pathology Laboratory, GSF, Munich, using standard protocols. The mice were under observation for early signs of leukemia. Aspiration of the blood and bone marrow was performed at 8 weeks and subsequently at four week intervals and assessed for engraftment of the mouse with transduced cells and progression of disease. [...] In the animal, the indications of initiation of disease were paleness of the feet, limited mobility or lethargy, short breaths or ruffled body hair. Moribund mice were sacrificed and analyzed as described.


Feuring-Buske, M., Frankel, A. E., Alexander, R. L., Gerhard, B., and Hogge, D. E. (2002). A diphtheria toxin-interleukin 3 fusion protein is cytotoxic to primitive acute myeloid leukemia progenitors but spares normal progenitors. Cancer Res 62, 1730-1736.

Pineault, N., Buske, C., Feuring-Buske, M., Abramovich, C., Rosten, P., Hogge, D. E., Aplan, P. D., and Humphries, R. K. (2003). Induction of acute myeloid leukemia in mice by the human leukemia- specific fusion gene NUP98-HOXD13 in concert with Meis1. Blood 101, 4529-4538.

Pineault, N., Helgason, C. D., Lawrence, H. J., and Humphries, R. K. (2002). Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol 30, 49-57.

5.10.7. BM transplantation and assessment of mice

For the bone marrow transplantation procedures, 8–10 week old recipient F1 (B6C3) mice were irradiated with 850 cGy from a 137Cs γ-radiation source. FACS purified transduced BM cells or defined ratios of transduced and unpurified and untransduced cells were injected into the tail vein of irradiated recipient mice.

PB or BM cell progeny of transduced cells were tracked using GFP fluorescence (Feuring Buske, 2002). Lineage distribution was determined by flow cytometric analysis as previously described (Pineault et al., 2003).

For histological analyses, sections of selected organs were prepared and H&E stained at the Academic Pathology Laboratory, GSF, Munich, using standard protocols. The mice were under observation for early signs of leukemia. Aspiration of the blood and bone marrow was performed at 8 weeks and subsequently at four week intervals and assessed for engraftment of the mice with transduced cells and progression of disease. The early signs of disease (leukemia) development were paleness of the feet, limited mobility or lethargy, shortness of breaths or ruffled body hair. Moribund mice were sacrificed and analyzed as described.


Feuring-Buske M., Frankel A. E., Alexander R. L., Gerhard B., Hogge D. E. (2002). A diphtheria toxin-interleukin 3 fusion protein is cytotoxic to primitive acute myeloid leukemia progenitors but spares normal progenitors. Cancer Res. 62 (6), 1730-6.

Pineault N., Buske C., Feuring-Buske M., Abramovich C., Rosten P., Hogge D. E., Aplan P. D., Humphries R. K. (2003). Induction of acute myeloid leukemia in mice by the human leukemiaspecific fusion gene NUP98-HOXD13 in concert with Meis1. Blood. 101 (11), 4529-38.

Anmerkungen

The source is not mentioned.

Note that there are two references "Pineault et. al., 2003" in the bibliography.

Sichter
(Hindemith) Schumann

[16.] Vpr/Fragment 037 01 - Diskussion
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3.13 Analysis of sacrificed/dead experimental mice.

Disease in the animals was determined by analyzing PB, spleen and BM of diseased sacrificed mice. PB was aspirated from heart with 1 ml insulin syringes; Heparin was used for anticoagulation. PB was used for blood smears on glass slides and RBC count. WBC count and immunophenotyping was performed after lysing the RBC with ammonium chloride. The size of the spleen in diseased mice was measured and the spleen was homogenized to single cell suspension in DMEM medium, for further analysis by FACS, cytospin or ex vivo assays. BM cells were obtained by flushing both femurs and tibias and subjected to WBC count, cytospin and immunophenotyping.

3.14 Preparation for histopathology.

The four limbs, tail and head of mice were pinned to a cork plate with needles after sacrificing the animals for histological analyses. Then the skin from the chin to the anus and also the abdominal muscles were opened in the median and also pinned (Fig.16). The mice were then bled by cutting one of the renal arteries, and the blood was sucked out with a tissue paper. Then the diaphragm was carefully cut away to allow the fixing liquid to get into the thorax. After this the whole mouse was placed into formalin over night, packed and sent to the histopathology, where the organs were cut and stained for analyses (Fig.16).

[page 93]

5.10.8. Analysis of sacrificed/dead experimental mice

The PB, spleen and BM of sacrificed mice were analyzed carefully. PB was aspirated from heart with 1 ml insulin syringes; Heparin was used for anticoagulation. PB was used for blood smears on glass slides and RBC count. WBC count and immunophenotyping was performed after lysing the RBC with ammonium chloride. The spleen size was measured in diseased mice, and then homogenized for single cell suspension in DMEM medium, for further analysis by FACS, cytospin or ex vivo assays. BM cells were obtained by flushing both femurs and tibias and subjected to WBC count, cytospin and immunophenotyping.

[page 94]

5.10.9. Preparation for histopathology

The four limbs, tail and head of mice were pinned to a cork plate with needles after sacrificing the animals for histological analyses. Then the skin from the chin to the anus and also the abdominal muscles were opened in the median and also pinned down (Fig.2.3). The mice were then bled by cut one of the renal arteries, and the blood was sucked out with a tissue paper. Then the diaphragm was carefully cut away to allow the fixing liquid to penetrate into the thorax. After this the whole mouse was placed into formalin (37%) over night, packed and sent to the department of pathology (PD. Dr. Leticia Quintanilla-Fend), where the organs were cut and stained for analyses.

Anmerkungen

The source is not mentioned here.

Sichter
(SleepyHollow02) Schumann

[17.] Vpr/Fragment 048 11 - Diskussion
Zuletzt bearbeitet: 2014-03-16 20:25:31 Graf Isolan
Fontanari Krause 2006, Fragment, KeineWertung, SMWFragment, Schutzlevel, Vpr, ZuSichten

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Seite: 48, Zeilen: 11-14
Quelle: Fontanari Krause 2006
Seite(n): 141, Zeilen: 10ff
Mice transplanted with BM cells expressing Cdx2 became moribund after a median of 90 days post transplantation (n=18) (Fig. 24). Diseased mice were characterized by cachexia, shortness of breath and lethargy, at which time they were sacrificed for further analysis. Mice transplanted with BM cells expressing Ostl became moribund after a median of 300 days post transplantation (n=3) (Figure 6.43). Diseased mice were characterized by cachexia, shortness of breath and lethargy, at which time they were sacrificed for further analysis.
Anmerkungen

Identical text adapted to the topic of the thesis.

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(Hindemith)

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