1. BCR-ABL1 Splice Patterns &
Generation of CML RNA bank
Placement Presentation
Deborah Reilly
7th May 2015
2. Paul O’Gorman Leukemia Research Centre
• Is a division of the University of Glasgow's Institute of Cancer Sciences
• Situated in the grounds of Gartnavel General Hospital which is located in the west end of
Glasgow
• Research and objectives:
- Centre’s research is focused around the understanding of the haematopoietic stem
cell (HSC)and its key role in the development of the vast variety of leukemic diseases
- Centre’s objective is to explore and identify therapeutic targets for leukaemia and in
turn produce new medication for patients
3. The Philadelphia chromosome, hallmark of CML,
results from translocation between chromosomes 9 and 22
Nature 1973, 243, 290 - 293
Bcr-Abl
# 9 # 22
c-Abl
Bcr
Ph1
Janet RowleyPeter Nowell
David Hungerford
Science 1960,132:1497
5. Project Outline (10 weeks)
• Set up/Learn techniques (2 weeks).
• Produce a bank of RNA/cDNA from:
(i) peripheral blood samples,
(ii) CML cell lines, and
(iii) primary CD34+ CML cells
• Determine baseline CFC
• Determine the BCR-ABL1 breakpoint using PCR
• Determine percentage CD34+CD38- by flow cytometry
• Use FISH to visualise the fusion oncogene
6. Functional Analysis of CML Stem/Progenitor Cells
Colony Forming Cells (CFC) assay
- Measures relatively mature, committed progenitor cells
CD34+ cells
Methylcellulose (H4034)
12-14 days
in culture
Total number of
colonies counted
8. PCR Changes
• The dNTPs dilution concentration was noticed to be x10 out – was amended
• The annealing temperature for the BCRABL1 F/R primers was calculated >60°C
The annealing temperature for the Actin 1/2 primers was calculated to be 58°C
- was running at 55°C , so had to be increased for BCRABL1
• Fresh cell line source as positive control
• An appropriate PCR programme on another thermal cycler was chosen
• Optimum PCR conditions discovered:
- For the BCRABL1 F/R primers:
- 3µM [Mg2+] addition with an annealing temperature of 65°C
- For the Actin 1/2 primers:
- 2µM [Mg2+] addition with an annealing temperature of 60°C
9. PCR Results
KY01
Cell lineages at their optimum PCR for primers BCRABL1 F/R and Actin 1/2:
b3a2 (13)
b2a2 (14)
Em2 Bv173 K562
KY01 NB4 Em2 Bv173
KY01
Splice variants of primary CD34+ CML samples:
KCL22 K562 CML 378
KY01 Em2 K562 CML 342
Were not
carried out in
the optimum
PCR conditions
Actin (housekeeping control)
10. Flow cytometry Results
• All of the primary samples were accessed for their viability and CD34+ CD38- expression using
flow cytometry
CML 404:
Q4 = 12.4%
11. FISH
• Probe properties:
- BCR = Green
- ABL = Red
- Fusion = Yellow
• Cell lineage K562 was used as the positive control:
- Larger than CD34+ stem cells
- Number of BCR:ABL signals roughly 3:2
- Have around 10 copies of the fusion oncogene per cell
• Primary CD34+ CML 416 sample:
- 1 BCR signal, 1 ABL signal and 2 fusion signals per cell
K562 CML416
In 1960 David Hungerford and Peter C. Nowell provided the first evidence for a genetic link to cancer when they identified an abnormally small chromosome in cells from patients with CML. They both worked in Philadelphia and therefore called this small chromosome the Philadelphia chromosome. In 1973, Janet Rowley saw, using Chromosomal banding techniques, that the Ph chromosome resulted from a reciprocal translocation between chromosome 9 and 22, and in the 1980s, the Ph chromosome was shown to carry a unique fusion gene, termed BCR-ABL, which is now believed to be the principal cause of CML.
