Slide #1.

Molecular Characterization of the Canine HMGB1 Presented by Melissa Metcalfe and Shauna Maguire
More slides like this


Slide #2.

Canine Genome An overview
More slides like this


Slide #3.

First Sequenced by Celera with the DNA from Shadow, a male Standard Poodle, in 2002. (WGS 80% coverage rough draft) Next major sequencing, done by the Broad Institute, was of Tasha, a 7 year old female boxer. (95% coverage)
More slides like this


Slide #4.

Genome Information       Genome Size 2.8x109 bp Gene sequences + EST’s 900 BAC end sequences 668 Sequence Tag sites (STS) 106 2n Chromosome number 78 38 autosomes + sex chromosomes
More slides like this


Slide #5.

Why sequence the canine genome?     Human genome and dog genome are evolutionarily and physiologically similar. The canine genome could help us understand analogous human hereditary diseases. Half of dog hereditary diseases have a human analog. Canines have been selectively bred for many years and their pedigrees have been kept. They are also have high levels of inbreeding. These factors make them ideal for genetic studies. Improvement of canine therapeutic treatments and purebred bloodline maintenance.
More slides like this


Slide #6.

HMGB1  High mobility group box protein 1  Originally identified as a transcriptional regulatory molecule that can modify chromatin structure by bending DNA.  Recently, it has been attracting considerable interest by oncologists because it has been said to have a “double life”.  What does it mean a “double life”?
More slides like this


Slide #7.

HMGB1     Besides its function as a transcriptional activator, HMGB1 has been found outside of the nucleus by secretion from macrophages. As a extracellular protein, HMGB1 participates in developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, and may be involved in the metastic invasion programme of cancer cells. Specifically, HMGB1 is a ligand for the receptor for advanced glycation end products (RAGE) thus activating p38mapk ,JNK, and p42/p44mapk which are key signaling pathways. This signal via a receptor induces inflammatory responses.
More slides like this


Slide #8.

Sepsis     Is the body’s systemic inflammatory response to infection or trauma. Can also be a caused by an infecting agent (such as bacteria). The body can mount a widespread inflammatory response to an infection that can quickly become out of control and cause even more harm. Can cause shock, organ damage, permanent disability, or death.
More slides like this


Slide #9.

Figure 1. Structure of the genomic elements and cDNA of the canine HMGB1.
More slides like this


Slide #10.

Table 1. Detailed analysis of the canine HMGB1 cDNA and genomic elements.
More slides like this


Slide #11.

Fig. 3 Comparison of Canine, Human, Mouse, and Bovine HMGB1 Protein
More slides like this


Slide #12.

Northern Blot Analysis Performed to define a basic expression pattern of the protein in can heart, lung, muscle, kidney, and spleen tissue. Lane Lane Lane Lane Lane Lane Lane Lane 1 2 3 4 5 6 7 8 – Kidney – Spleen – Spleen – Heart – Heart – lung – Muscle - Fibroblasts
More slides like this


Slide #13.

Fluorescent In Situ Hybridization Makes it possible to map any cloned locus to its position on the metaphase chromosome. Good for quickly obtaining the chromosomal position of a newly cloned locus. Does not require mapping to be “in relation” to another.
More slides like this


Slide #14.

FISH protocol     Drop cells onto glass slide Gently denature DNA with DNase Hybridize with fluorescent probe and wash away unhybridized probe Expose to UV light and take a picture of the fluorescent chromosome with a Fluorescence microscope.
More slides like this


Slide #15.

Metaphase spread after FISH with signals on both chromosomes (a) and after GTGbanding (b).
More slides like this


Slide #16.

Conclusion    “The molecular characterization of the canine HMGB1 gene and protein showed that humans and dogs share an identical HMGB1 protein.” Molecular targeting of HMGB1 in dogs is very significant for therapeutic approaches in humans due to the similarity of genesis and development of diseases in both species. Thus, the canine shows great potential as a model organism in the study of human diseases.
More slides like this