1. In Vivo Expression of Murine Platelet Glycoprotein Ibα
by Hiroyuki Fujita, Yoshimi Hashimoto, Susan Russell, Barbara Zieger, and Jerry Ware
Blood
Volume 92(2):
July 15, 1998
©1998 by American Society of Hematology

2. Genomic arrangement of the mouse GP Ibα gene.
Genomic arrangement of the mouse GP Ibα gene. Restriction enzyme analysis of a P1 plasmid containing a fragment of mouse genomic DNA identified contiguous BamHI andBamHI/EcoRI restriction fragments that were chosen for DNA sequence determination. Based on sequence alignment with the human GP Ibα gene, boundaries for two exons are proposed (boxed regions) with an open reading frame (shaded box) encoding the putative mouse GP Ibα precursor polypeptide.19 A shaded gray region under the schematic representation of the gene identifies the approximate position of a radiolabeled fragment used for Northern analysis (Fig 2). The nucleotide sequence has been deposited in GenBank (accession no.U91967).
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

3. Northern blot analysis of RNA prepared from the major murine organs.
Northern blot analysis of RNA prepared from the major murine organs. Total RNA was isolated from nine organs of adult mice and electrophoresed through a 1% agarose/formaldehyde denaturing gel. After electrophoresis, the RNA was transferred to a nitrocellulose membrane and hybridized with a radiolabeled probe of the murine GP Ibα coding sequence.19 A representative photograph of the autoradiograph obtained after hybridization and washing documents an RNA species of 2.7 kb in RNA prepared from bone marrow of a mouse femur. The size of the RNA is consistent with the predicted size of the transcript encoding mouse GP Ibα (Fig 1). No other hybridizing signals were observed after a lengthy (2 weeks) exposure to x-ray film. The migrating position of an RNA molecular weight standard is shown to the left. After obtaining the autoradiograph, the nitrocellulose membrane was stripped of radioactivity and rehybridized with a radiolabeled DNA probe from the mouse 18S rRNA gene to confirm similar amounts of RNA were loaded from the different RNA preparations (lower panel).
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

4. Promoter alignments of mouse and human GP Ibα gene sequences.
Promoter alignments of mouse and human GP Ibα gene sequences. Mouse (Mo) and human (Hu) GP Ibα gene sequences are aligned flanking the transcription initiation site of the human GP Ibα gene (nucleotide +1). Negative nucleotide numbering relative to the transcription initiation site is shown to the left of each sequence. The human GP Ibα gene is composed of a 5′ untranslated exon (exon I), a single intron, and a single exon (exon II) containing the initiating methionine codon (ATG). The exons are highlighted by a shaded box with only a limited 5′ portion of exon II displayed. Mouse exon I corresponds to nucleotides 2,387 to 2,466 and exon II begins at nucleotide 2,659 of GenBank accession no.U The human 5′ sequence contains GATA and Etscis-acting elements, which have previously been shown by mutagenesis to be essential for promoter activity in megakaryocytic-like cell lines.20 Mutated bases of the human sequence that eliminated promoter activity are highlighted by black boxes at nucleotides −150 to −142 (Ets) and −92 to −91 (GATA).20 The mouse GP Ibα sequence displays a similar overall arrangement with conserved GATA and Ets elements along with positive regulatory element (MegPos) identified in the rat and human platelet factor 4 promoters.41 Translated sequence for the first 14 residues of the human and mouse GP Ibα signal peptides is shown in exon II with a single-letter notation for each residue except where there exists sequence differences, in which case both species-specific amino acids are shown. A BamHI restriction site is underlined (nucleotides ) and corresponds to the 3′ boundary of the promoter fragment used to generate transgenic mice expressing the reporter protein, luciferase. Human GP Ibα DNA sequence corresponds to GenBank accession no. M22403.
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

5. Northern blot analysis of RNA prepared from transgenic murine organs.
Northern blot analysis of RNA prepared from transgenic murine organs. Transgenic mice were generated expressing the reporter protein, luciferase, under the control of a BamHI promoter fragment of the murine GP Ibα gene (Fig 1). Five mouse colonies were expanded from individual founder mice. Results are presented from one mouse line and are typical of each of the five lines in which expression of the transgene was observed. As described in Fig 2, total RNA was prepared from the major organs of the transgenic mice and analyzed by Northern analysis. The luciferase mRNA transcript of 2.4 kb was detected using a radiolabeled fragment from the coding sequence for luciferase. Similar to results probing for the endogenous GP Ibα transcript, a transcript is only visible in RNA prepared from the marrow of a mouse femur. Subsequent hybridization of the same filter with a portion of the mouse 18S rRNA gene is shown to illustrate similar RNA levels for each lane.
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

6. Luciferase activity in transgenic mouse colonies.
Luciferase activity in transgenic mouse colonies. Luciferase activity in organ homogenates from two transgenic colonies (32 [n = 5] and 12 [n = 5]) derived from independent founders is presented. The mean and standard error of luciferase activity (RLUs per μg of protein) are shown. Maximal luciferase activity was observed in bone marrow and spleen, both hematopoietic organs in mice and both containing murine megakaryocytes. Luciferase activity was also detected in blood, but is not apparent in this figure owing to the small contribution of platelet protein to the total protein found in whole blood (see Results). Consistent, albeit low levels, of luciferase activity were observed in lung, heart, and aorta even after extensive perfusion of the mouse. The relevance of low levels of gene activity in these organs is discussed.
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

7. Luciferase activity in transgenic platelets.
Luciferase activity in transgenic platelets. A linear correlation exists between the number of platelets and luciferase activity in the transgenic mice. PRP and PPP from transgenic mice (colony 32) was used to resuspend blood cells and subsequently determine luciferase activity. These reconstitution experiments confirmed the luciferase activity in whole blood coincides completely with the presence of platelets with a linear correlation between luciferase activity and the number of platelets (r 2 = 0.88).
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

8. Transgene activity in an animal model of gram-negative sepsis.
Transgene activity in an animal model of gram-negative sepsis. LPS was administered to mice (colony 32) containing a luciferase transgene under the control of the mouse GP Ibα promoter. The wide range of LPS-induced effects and toxicity in mice is well documented and doses administered to individual mice were sufficient to achieve maximal levels for a variety of cytokines. LPS was administered by intraperitoneal injection (25 mg/kg) and luciferase activity in the major murine organs was determined. Results are shown for assays performed 24 hours postinjection, although similar results were obtained at 4-hour intervals leading up to the 24-hour assay shown. The mean and standard error of the mean are shown for each organ (n = 4). Control mice were injected intraperitoneally with an equal volume of saline buffer. No differences in the levels of luciferase activity were observed at any time point.
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology

9. Northern analysis of GP Ibα expression in mice administered LPS
Northern analysis of GP Ibα expression in mice administered LPS. As a model of gram-negative sepsis, LPS was administered to mice (colony 32) by intraperitoneal injection (25 mg/kg).
Northern analysis of GP Ibα expression in mice administered LPS. As a model of gram-negative sepsis, LPS was administered to mice (colony 32) by intraperitoneal injection (25 mg/kg). As described in Fig 2, total RNA was prepared from treated (+) and control (−) mice. (A) Lanes 1 to 9 correspond to the same organs listed in the legend for Fig 2. Again, a visible GP Ibα transcript is only present in bone marrow (lane 9) with no increase in GP Ibα expression detected in any organ, but an absence of the GP Ibα bone marrow transcript 24 hours post-LPS treatment. (B) The nitrocellulose filter of (A) was rehybridized with an ICAM-1–radiolabeled cDNA fragment and confirmed an inflammatory state in the treated mice with an increase in the lung ICAM-1 mRNA (lane 5).
Hiroyuki Fujita et al. Blood 1998;92:
©1998 by American Society of Hematology