Transgenic zebra fish embryo model for hematopoiesis and lymphoproliferative disorders

Active Publication Date: 2005-09-08
PARKER HUGHES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0013] In an alternative embodiment of the invention, the ZF embryo is transformed with a non-DNA binding form of Ikaros. The non-DNA binding form can be, for example, Ik-4, 5, 6, 7, 8, 9, or 10, each of which lacks the three N-terminal zinc fingers required to confer high affinity DNA binding. A mutant Ikaros protein can al

Problems solved by technology

Thus, splicing errors can have severe consequences for the lymphocyte compartment of the developing immune system.
An abundance of dominant-negative Ikaros isoforms that no long

Method used

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  • Transgenic zebra fish embryo model for hematopoiesis and lymphoproliferative disorders
  • Transgenic zebra fish embryo model for hematopoiesis and lymphoproliferative disorders
  • Transgenic zebra fish embryo model for hematopoiesis and lymphoproliferative disorders

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example 1

Expression Constructs and Microinjection of Zebrafish Embryos

[0052] Fish and embryos. The adult wild type ZF were maintained generally according to the Zebrafish book recommendations (Westerfield, 1995). Males and females were kept in 10 G tanks, 70 fish per tank, with a constant slow flow of conditioned water at 26° C. and a controlled 14 hours day / 10 hours night cycle. Embryos were obtained through natural spawning in breeding cages with a netted false bottom or by in vitro fertilization using eggs and milt collected from the mature females and males anesthetized with tricaine (Sigma). Embryos were kept at 28.5° C. in Petry dishes, 30-50 per dish.

[0053] Five days after hatching, frys were transferred to a nursery for 2 weeks and raised in 1 G mouse cages at 28.5° C. Larvae were fed with live food, paramecia and brine shrimps, according to recommendations of Dr. Stephen Ekker (personal communication). The survival rate was over 95%. Juvenile ZF were transferred to 10 G tanks, tre...

example 2

Transgenic Expression of Human Ikaros Isoforms Ik-4 and Ik-2 in ZF Embryos

[0068] Linearized expression vectors hIK4 wt pFV4aCAT and hIK2wt pFV4aCAT were mixed with GFP mRNA and microinjected into one-cell stage ZF embryos to force expression of the dominant-negative human Ikaros isoform Ik-4 and the DNA binding human Ikaros isoform Ik-2 (control) during primitive hematopoiesis. The microinjections were successful in >95% of all embryos, as evidenced by a strong green fluorescence documenting the expression of the coinjected GFP mRNA from mid-gastrula until prim-5 stage (FIGS. 1A1, 2A1, 3A1).

[0069] Total mRNA was extracted from GFP-positive individual ZF embryos at 6 hpf, mid-gastrula stage (FIG. 1A1), at 24 hpf, prim-5 stage (FIGS. 1B1, 1C1) and at 48 hpf, Long-pec stage (FIG. 1D1). The extracted mRNA was reverse-transcribed using oligo-dT and random hexamers. The resulting cDNAs were amplified with ZF β-actin primers (FIGS. 1A3, 1B3, 1C3, and 1D3) to test the integrity of the ext...

example 3

In Situ Localization of nIkaros Transgenes

[0073] The topographical profile of the human Ikaros transgenes Ik-4 and Ik-2 expressed in the ZF embryos at 17-19 hpf was confirmed by whole-mount in situ hybridization using digoxigenin-labeled Ikaros riboprobes. Probes hybridized to the human Ikaros mRNA were immunolocalized with anti-DIG Fab fragments and detected by chromogenic reaction with NBT / BCIP. No false positive signals were detected in non-transgenic control ZF embryos (FIG. 2A). In transgenic ZF embryos, the chromogenic (blue-purple) signal of human Ik-4 or Ik-2 transgene expression was largely localized to the trunk region containing the intermediate cell mass (ICM) where primitive hematopoiesis takes place (FIGS. 2B-2F), reminiscent of the expression profile of other regulators of hematopoiesis such as GATA-1 and c-MYB (Detrich et al., 1995, Amatruda and Zon, 1999).

[0074] At 48 hpf, embryonic hematopoiesis in the ZF shifts from the ICM to the dorsal mesentery and forms the ...

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Abstract

A transgenic zebrafish animal model for the study of haemopoietic cell differentiation, control, and screening of therapeutic agents.

Description

BACKGROUND OF THE INVENTION [0001] Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children (Pizzo and Poplack, 1993, Greaves, 1986, Uckun et al., 1998, Crist et al., 1988). A better understanding of the biological basis and predisposing leukemogenic events in this disease is needed in order to develop more effective treatment programs as well as novel prevention strategies. [0002] Leukemic clones are thought to originate in ALL patients from normal lymphocyte precursors arrested at various stages of T- or B-lymphocyte development (Greaves, 1986). Accordingly, any critical regulatory network that controls normal lymphocyte development is a potential target for a leukemogenic event. [0003] One such regulatory network vital for normal hematopoiesis involves Ikaros, a member of the Kruppel family “zinc finger” DNA-binding proteins. Ikaros acts as an evolutionarily conserved “master switch” of hematopoiesis that dictates the transcriptional regulation of lymphocy...

Claims

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Application Information

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IPC IPC(8): A01K67/027C07K14/47C12N15/85
CPCA01K67/0275A01K2207/15A01K2217/00A01K2217/05A01K2217/072C12N2830/008A01K2267/0331A01K2267/0381C07K14/4705C12N15/8509A01K2227/40
Inventor UCKUN, FATIHBENYUMOV, ALEXEY
Owner PARKER HUGHES INST
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