Download Telomere Shortening and Tumor Formation by Mouse Cells Lacking

Telomere Shortening and Tumor
Formation by Mouse Cells Lacking
Telomerase RNA
Maria A. Blasco et al. (1997), Cell Vol. 91,
p. 25 - 34
Outline
1. Introduction
2. Objective
3. Results
4. Summary
5. Take-Home-Lesson
6. Discussion
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Introduction
Telomeres:
-at the end of chromosomes
-repetitive DNA
(vertebrates: (TTAGGG)n )
-contain non-coding DNA
material
-protect chromosomes
-prevent fusions and
degradation and a constant
loss of important DNA from
chromosome ends
http://www.wissenschaft-online.de/sixcms/
media.php/912/thumbnails/Telomer1.jpg.644586.jpg
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Introduction
Problem:
the chromosomes shorten with
every cell divison
Reason:
DNA-Polymerase moves in the 5‘
to 3‘ direction and needs RNAPrimer to start the building of
okazaki fragments (for lagging
strand).
Replication is possible as long as
RNA-Primer can bind to the
http://upload.wikimedia.org/wikipedia/commons/9/93/Dn
areplication.png
template stand
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Introduction
Solution: enzyme telomerase
-ribonucleoprotein with reverse transcriptase activity
→ RNA – component serves as a template for the telomere
repeats
(template region of telomerase is 3'-CAAUCCCAAUC-5’)
→ adds DNA sequence repeats (5’-TTAGGG-3’ in all
vertebrates) to the 3’ end of DNA strands in the telomere
region
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Introduction
Telomerase binds to 3‘ end
of the leading strand, that is
complementary to the
telomerase RNA
RNA-component is used as
template
→ adding of repeating
sequence (5‘-TTAGGG-3‘)
is possible
the DNA-Polymerase
complements the lagging
strand
http://en.wikipedia.org/wiki/File:
Working_principle_of_telomerase.png
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Objective
The objective is to describe the effects of the absence of
telomerase activity on telomere length, cellular viability,
neoplastic transformation and tumor formation in nude mice.
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Results
Goal 1: Generation of a knockout mouse
The telomerase RNA component (mTR) is necessary for
telomerase activity
→ RNA component functions as template
To generate a mouse deficient for telomerase activity, the
mTR gene was deleted from the mouse germline
Therefore they constructed the plasmid pPNT-mTRΔ, which
allows replacement of the mTR gene with the neomycin
resistance gene (homologous recombination)
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Results
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Results
Positive and negative selection of ES cells
When homologous recombination was successful, the ES
cells are resistant against
● Neomycin → antibiotic resistance
● Ganciclovir → loss of the marker HSV thymidine kinase
(HSV-TK)
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Results
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Results
Southern Blot:
1: mTR+/+ wildtype
2: mTR+/- heterozygous
3: mTR-/- homozygous
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Results
Observation:
The generated mTR-/- mice were able to live
→ mTR is not essential for embryonic development
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Results
Goal 2: Determination of fertility
Crossing first generation G1 mTR-/- mice to each other
→ G2 mTR-/- mice were obtained
→ mTR is not essential for fertility
For the 6 generations, which were analyzed, all mice were
alive. To get a new generation, they always crossed two mice
from the previous generation to each other
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Results
Goal 3: Detection of telomerase activity in mTR-/- mice and
mouse embryonic fibroblasts (MEF)
MEF cultures and S100 extracts from brain, liver, thymus
and spleen were tested for telomerase activity with the
TRAP assay
TRAP assay is based on the PCR amplification of a
substrate, which is extended by telomerase
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Results
PCR for the MEF cultures
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Results
Observation:
Telomerase activity was detected in mTR+/+ and mTR+/MEF cultures and S100 extracts
→ TRAP assays showed the absence of telomerase
activity in mTR-/- embryonic fibroblasts and adult mice
tissues
→ mTR gene is essential for mouse telomerase
activity in vivo
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Results
Goal 4: Recovering of telomerase activity of mTR-/- MEFs
mTR gene was
reintroduced into mTR-/MEF cultures by transient
transfection
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Results
Observation:
48 h after the transfection the MEF cultures had telomerase
activity
The cells, transfected with an empty plasmid, had no
telomerase activity
→ telomerase RNA is responsible for the telomerase
activity.
→ mTR-/- MEFs are able to express telomerase activity
after reintroduction of the mTR gene into the cells
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Results
Goal 5: Examination of the growth of mTR-/- MEF cultures
MEF cultures were generated from mTR+/+ , mTR+/- and
mTR-/- G1 embryos and from mTR-/- G2, G3, G4 and G6
embryos
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Results
Observation:
● the growth rates of all MEFs were similar
→ missing telomerase activity does not inhibit the cell
division
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Results
Goal 6: Determination of telomere length in mTR-/- cells
MEF cultures were derived from G1 wildtype embryos and from
mTR-/- G1 - G6 embryos
Southern Blot:
Genomic DNA was blotted and
probed with a TTAGGG-probe to
detect terminal fragments
→ it is not obvious, that telomeres of
mTR-/- generations are shorter
than the ones of the wildtype
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Results
Same treatment with MEF cultures from G1 wildtype and from
mTR-/- G1 – G4 animals but this time DNA was isolated from
cells during early, mid and late passage
→ telomere fragments
shorten with increasing cell
doublings in culture, when
telomerase is absent
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Results
Fluorescence in situ hybridization (FISH)
MEF cultures from G1 WT and mTR-/- G2, G4 and G6 animals
→ all chromosome ends were examined with a (TTAGGG)probe, which had a fluorescence-tag
Definition:
1 telomere fluorescence unit (TFU) represents 1 kb of
(TTAGGG)-repeats
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Results
Observation:
The TTAGGG-signal
decreases with
increasing mTR-/generations
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Results
FISH images:
In G6 cultures there was
no TTAGGG-signal for
5% of the chromosomes
end-to-end-associations
of chromosomes were
observed more frequently
→ no telomerase activity
leads to instability
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Results
Goal 7: Examination of tumor formation in the absence of
telomerase activity
MEF cultures from
- G1 mTR+/+
- G1 mTR+/- G1, G2, G3, G4 and G6 mTR-/- mice
Infection with retrovirus, carrying the genes for adenovirus
E1A, activated protooncogene rasv12 and puromycin drug
resistance
→ oncogenic transformation
→ foci were selected and 105 cells were injected in nude
mice
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Results
Tumors were generated from the mTR+/+ , mTR+/- and all
of the mTR-/- cells
→ telomerase activity is not essential for transformed cells
to form tumors in nude mice
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Summary
• the
telomerase RNA component (mTR) is required for
telomerase activity
• telomerase activity is not necessary for embryonic
development or fertility
• the absence of telomerase has no influence on the growth
rates of cultures
• Without telomerase, the telomere length decreases from
generation to generation and also during cell doublings in
culture
• telomerase is not responsible for tumor formations
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Take-Home-Lesson
Telomerase is necessary for telomere length conservation
but is not essential for establishment of cell lines,
oncogenic transformation or tumor formation.
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Discussion
1. Wie werden Knockout-Mäuse erzeugt?
Beschreiben sie stichwortartig das Vorgehen.
2. Welche Aufgabe hat die Telomerase?
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Welche Aufgabe hat die Telomerase?
Die Telomerase ist ein Ribonukleoprotein und wirkt als reverse
Transkriptase, wobei die RNA-Komponente als Matrize bei der
Verlängerung der Telomere dient.
→Telomerase verlängert Telomere und wirkt somit einer Verkürzung
der Chromosomen bei der Replikation entgegen
→Telomerase verhindert den Verlust essentieller genetischer
Information nach jeder Replikationsrunde
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