Paul O'Gorman placement presentation
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Transcript of Paul O'Gorman placement presentation
BCR-ABL1 Splice Patterns & Generation of CML RNA bank
Placement PresentationDeborah Reilly7th May 2015
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
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 NowellDavid Hungerford
Science 1960,132:1497
2 11
11
5’ 3’
23
mBCR
13 14
MBCR
15 16 17 18 19 20 21 22
mBCR
1 2 3 4 5 6 7 8 9 10 11 12
5’ 3’
1b 1a 2 3 4 5 6 7 8 9 10 11
c-ABL gene (chromosome 9)
BCR gene (chromosome 22)
13
2 11
p230bcrabl
c-ABL geneBCR gene
1
21 13
1 19
p190bcrabl (e1a2)
p210bcrabl (e13a2, e14a2)
13
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
Functional Analysis of CML Stem/Progenitor Cells
Colony Forming Cells (CFC) assay
- Measures relatively mature, committed progenitor cells
CD34+ cellsMethylcellulose (H4034)
12-14 days in culture
Total number of
colonies counted
CFC Results
CML 404:
BFU-E
BFU-E
CML 342:
BFU-E + CFU-GM
CML 361:
CFU-GM
CML 378:
BFU-E
BFU-E + CFU-GM
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
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)
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%
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
Table of Results + RNA/cDNA bank
UPN % Recovery Actin BCR-ABL1 breakpoint CFC /100 cells RNA (ng/µL) 260/280 % CD34+ CD38-
308 0.5 - - - - - 35.5
339 84 - TBD 1.78 90.15 2.05 36.4
342 130 b2a2 (13) 1.43 158.63 1.97 17.1
361 15 - TBD 0.49 2.7 1.85 36.9
372 70 ND 0.77 43.04 1.92 13.0
378 84 b3a2 (14) 1.08 23.65 2.18 13.0
379 44 - TBD 0.16 3.78 2.32 ND
381 50 b3a2 (14) 0.29 7.01 2.11 32.4
404 62 - TBD 1.67 60.87 2.02 12.4
416 48 - - - - - -
SAMPLE RNA cDNA
CML 308 x x
CML 339
CML 342
CML 361
CML 372 x
CML 378
CML 379 x
CML 381
CML 404
CML 416 x x