Karl, a new Drosophila Lipocalin gene, specifically expressed in the blood cell compartment.

1
Karl, a new Drosophila Lipocalin gene,
specifically expressed in the blood cell
compartment.

• Ulla-Maja Bailey, PhD.
• Stockholm University
• WGI Developmental Biology

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Background

• What is the function of blood cells in
  Drosophila? -Immunity, -Apoptosis, -Metamophosis,
  -etc.
• What do we know about the blood cells in
  Drosophila? Plasmatocytes, Crystal cells and
  Lamellocytes.
• There are homologies to mammalian differentiation
  of blood cells. Both requier members of the GATA
  transcription factor family.

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Prohemocytes arise from a discrete subpopulation
of mesodermal cells located in the head of the
embryo. Lebestky et al.(2000) demonstrated that
hemocyte precursor cells express the GATA factor
srp. The more mature cells starts to express Lz
becoming Crystal cells or Gcm becoming
Plasmatocytes with macrophage like properties.
In the larvae mature hemocytes are released from
the Lymph Gland (LG). The majority of the
circulating hemocytes are small plasmatocytes.
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New molecular markers for Drosophila blood cells

• Generate a cDNA library from Drosophila blood
  cells.
• Generate a cDNA microarray chip based on the
  library.

mm
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Mutants with blood cell defects
Overproliferation or fewer blood cells.
Control
Mutant
mRNA
mRNA
cDNA
cDNA
Cy3
Cy5
mix
Hybridize on chip
Select clones for sequencing
In situ hybridization
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The hematopoetic organs
In situ with clone 14
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Cloning of the Karl gene

• The cDNA is 1444bp long
• Open reading frame encoding a putative protein of
  266 amino acids length. Predicted 31kDa size.

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Northern blot assay

• Karl is expressed at a low level throughout
  development.
• In the Tum-l mutant and in the blood cell line
  mbn-2 expression of Karl is stronger.

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Future plans

• Extensive in situs and RT-PCR studies.
• Knock-out Karl in mbn-2 cells with dsRNA.
  -phagocytosis and other immune reactions.
• Generate antibody against Karl. -Intracellular
  localization.
• Purification and binding assays.