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Immortalization of Primary Human Cells with Telomerase
|Immortalization of Primary Human
Cells with Telomerase
One major obstacle to the immortalization of
primary human cells is telomere-controlled senescence.
Telomere-controlled senescence is caused by
the shortening of the telomeres that occurs each time
somatic human cells divide. The enzyme telomerase
can prevent the erosion of telomeres and block the
onset of telomere-controlled senescence, but its expression
is restricted to the human embryo and, in the
adult, to rare cells of the blood, skin, and digestive
track. However, we and others have shown that the
transfer of an exogenous hTERT cDNA, encoding the
catalytic subunit of human telomerase, can be used to
prevent telomere shortening, overcome telomerecontrolled
senescence, and immortalize primary
human cells (Bodnar et al.
, 1998). Most importantly,
hTERT alone can immortalize primary human cells
without causing cancer-associated changes or altering
phenotypic properties (Jiang et al.
, 1999; Morales et al.
1999; Ouellette et al.
, 2000). Primary human cells that
have been immortalized successfully with hTERT
alone include fibroblasts, retinal pigmented epithelial
cells, endothelial cells, myometrial cells, esophageal
squamous cells, mammary epithelial cells, keratinocytes,
osteoblasts, and Nestin-positive cells of the
pancreas (Bodnar et al.
, 1998; Yang et al.
, 1999; Ramirez et al.
, 2001; Yudoh et al.
, 2001; Condon et al.
, 2002; Lee et al.
, 2003; Morales et al.
, 2003). This article describes
the use of hTERT for the purpose of immortalizing
primary human cells using the primary human fibroblast
as an example.
II. MATERIALS AND
Packaging cell line Phoenix-ampho (ΦNX-A) for the
production of amphotropic viruses is available from
Dr. Gary Nolan (Stanford University, Stanford, CA;
vectors pBabePuro and pBabePuro-hTERT are from
Geron Corp. (Menlo Park, CA; Ouellette et al.
Dulbecco's modified Eagle's medium (DMEM; Cat.
No. 11995), medium 199 (Cat. No. 11150), gentamicin
(Cat. No. 15710), and trypsin-EDTA (0.05% trypsin,
0.53 mM EDTA; Cat. No. 25300) are from Invitrogen
Corp. (Carlsbad, CA). Electrophoresis apparatus (Cat.
Series 21070), including glass plates, spacers, and
combs, are from Life Technologies (Rockville, MD).
Cosmic calf serum (Cat. No. SH30087) and fetal bovine
serum (Cat. No. SH30070) are from HyClone (Logan,
UT). The polysulfone filter (0.45µm, Cat. No.
DD50402550) is from Life Science Products Inc.
(Frederick, CO). Dimethyl sulfoxide (DMSO; Cat. No.
D 2650), polybrene (Cat. No. H 9268), puromycin
(Cat. No. P 7255), and bovine serum albumin (BSA; Cat.
No. A 2153) are from Sigma-Aldrich (St. Louis, MO).
The MBS mammalian CaPO4
transfection kit is from
Stratagene (Cat. No. 200388; La Jolla, CA). The TrapEZE
kit is from Serologicals Corp. (Cat. No. S7700; Norcross,
-Tetramethylenediamine (TEMED; Cat.
No. 161-0800) and 40% acrylamide:bisacrylamide
[19:1] solution (Cat. No. 161-0146) are from Bio-Rad
(Hercules, CA). The PhosphorImager is from Molecular
Dynamics (Model No. 810; Sunnyvale, CA). Nalgene Cryo 1°C freezing containers are from Nalge Nunc
International (Cat. No. 5100-0001; Rochester, NY). T4
polynucleotide kinase is from New-England BioLabs
(Cat. No. M0201S; Beverly, MA). [γ-32
P]ATP is from
New England Nuclear (Cat. No. 502A12070; Boston,
MA). The polymerase chain reaction (PCR) is performed
using a PCRExpress thermocycler from
Hybaid (Ashford, Middlesex, UK). All other chemicals
are from Fisher Biotechnology (Fait Lawn, NJ), Fisher
Scientific Co. (Pittsburgh, PA), or Sigma-Aldrich (St.
A. Production of Replication-Defective
Retroviruses Carrying the hTERT cDNA
Primary human cells tend to transfect very poorly.
Consequently, the preferred method of transferring
exogenous telomerase to such cells is their transduction
with replication-defective retroviruses carrying an
hTERT cDNA. The following procedure, modified
from Pear et al.
(1993), yields high-titer viruses following
the transient transfection of ΦNX-A cells, a retroviral
packaging cell line.
- 293T medium: To 500 µl of DMEM, add 50 ml of fetal
bovine serum and 500µl of 10mg/ml gentamicin.
Store at 4°C and protect from light.
- Complete medium X: To a clean sterile 500-ml
bottle, combine 400 ml of DMEM, 100 ml of medium
199, 50ml of cosmic calf serum, and 500µl of
10mg/ml gentamicin. Store at 4°C and protect from
B. Transduction of Human Primary Cells
with Retroviruses Carrying hTERT
- In a 37°C water bath, thaw a vial of ΦNX-A cells
and transfer cells to a culture dish containing 293T
medium. Incubate cells at 37°C under 5% CO2. The
next day, replace medium with fresh medium.
- Expand ΦNX-A cells in 293T medium using split
ratios of I : 4 to 1:6. Avoid letting the cells grow beyond
90% confluence. To split cells, remove medium by aspiration,
gently wash cells once with PBS. Take extra care
not to dislodge the cells, as ΦNX-A cells tend to detach
very easily. Add 2ml of trypsin-EDTA per 100-mm
dish and let cells detach at 37°C for 1-2min. Tap dish
to help dislodge cells, add 5 ml of 293T medium, and
pipette up and down to break cell clusters. Transfer
suspension to a sterile tube and pellet cells by lowspeed
centrifugation (2000g for 5 min at room temperature). Remove supernatant and resuspend cells in
5 ml of 293T medium.
- Following the manufacturer's instructions, count
cells using a Coulter counter or hemacytometer. In two
100-mm plates containing 293T medium, seed 5.0 × 106 cells/dish. Freeze remaining cells to replenish archival
stocks, if needed (refer to Section III,C).
- On the following day, ΦNX-A cells should be
~80% confluent and ready to be transfected with the
retroviral vectors. Transfect each dish with 10-20µg
of plasmid; one with vector pBabePuro-hTERT and
the other with the empty vector, pBabePuro. ΦNX-A
cells are transfected most easily using the CaPO4
method, but other methods can be used as well. To
CaPO4 transfect the ΦNX-A cells, we have used the
Stratagene's MBS mammalian transfection kit following
the manufacturer's instructions. In a 5-ml Falcon
2054 polystyrene tube, prepare a calcium-DNA
precipitate by mixing plasmid DNA (10-20 µg in 450 µl
of sterile water) with 50µl of solution I (2.5M CaCl2)
and then adding 500µl of solution II [N,N-bis(2-
hydroxyethyl)-2-aminoethanesulfonic acid in buffered
- Incubate the calcium-DNA mixtures at room
temperature for 10-20min. In the meantime, prepare
the cells by washing cells once with PBS and feeding
them with 10ml/dish of 293T medium, in which 6%
modified bovine serum (provided by the kit) is used
in place of the fetal bovine serum.
- Resuspend the DNA precipitate gently and then
apply dropwise in a circular motion to the dishes, as
to distribute the DNA evenly. Swirl dishes once.
- Incubate cells at 37°C under 5% CO2 for 3 h.
- Remove medium by aspiration.
- Wash cells once with sterile PBS and feed with
293T medium. Return cells to 37°C under 5% CO2.
Cells have now been transfected and should start producing
retroviruses within the next 24 h.
- Viruses can be collected in three consecutive
harvests over the next 24-72h posttransfection. To
collect viruses, replace the 293T medium with 4-5 ml
of the target cells culture medium, in which the viruses
will now be allowed to accumulate. For primary
human fibroblasts, collect viruses in complete medium
X. Return cells to 37°C under 5% CO2 with the dishes
spread on a leveled shelf to ensure good coverage of
- After 8-16h of exposure to the cells, harvest
supernatants and then force through a 0.45-µm polysulfone
filter so that all remaining cells are removed.
Bleach and discard the transfected cells once the last
batch of viral supernatant has been collected.
- Keep viral supernatants either on ice and use
within the hour or else aliquot, freeze, and store at -80°C where they can last for approximately 6
months. Label each aliquot with name of retroviral
vector, harvest medium, date, and harvest number
(1st, 2nd, etc...). Freezing does not appear to cause
substantial drops in titer, but cycles of freeze/thaw do.
- 400µg/ml polybrene: Dissolve 8 mg of polybrene in
20ml of deionized double-distilled water. Sterilize
by filtration through a 0.45-µm polysulfone filter.
Store at 4°C.
- 500µg/ml puromycin: Dissolve 10rag of puromycin
in 20ml of deionized double-distilled water. Sterilize
by filtration through a 0.45-µm polysulfone
filter. Store aliquots at-20°C.
C. Other Procedures Related to
the Immortalization of Human
1. Freezing ΦNX-A Cells and Primary Human Cells
- Thaw primary human fibroblasts in complete
medium X. Incubate cells at 37°C under 5% CO2. The
next day, replace medium with fresh medium.
- Expand cells in complete medium X using split
ratios of 1:2, 1:4, and 1:8 for late, mid, and early
passage cells, respectively. Avoid leaving monolayers
at full confluence for extensive periods of time, as
cells may then resist trypsinization. To split cells,
remove medium and wash cells twice with 2ml of
trypsin-EDTA per 100-mm dish. Incubate dishes at
37°C until cells have detached (1-3 min). Tap dishes to
help dislodge cells, add 5 ml of complete medium X,
and pipette up and down to dissociate clumps. Transfer
suspension to a sterile tube and pellet cells by
low-speed centrifugation (2000g for 5min at room
temperature). Remove supernatant and resuspend
cells in 5ml of complete medium X. While keeping
track of population doublings (see later), expand
fibroblasts until sufficient cells are available.
- Seed four wells of a 6-well plate with target cells
so that the cells are in log-phase growth on the next
day. For primary human fibroblasts, seed cells in complete
medium X at 2 × 105 cells per well. Take note of
the population doubling of the seeded cells as the
initial PD (or PDi). Freeze remaining cells to replenish
archival stocks, if needed. Label frozen vials with
name of strain, date, population doubling, and
approximate number of cells per vial (refer to Section
- Incubate dishes overnight at 37°C under 5% CO2.
- Remove the medium from all dishes. Infect one
dish with viruses carrying hTERT (sample A; infected
with pBabePuro-hTERT) and a second dish with control viruses carrying no insert (sample B; infected
with pBabePuro). Feed the remaining two dishes with
virus-free medium (samples C and D; uninfected).
Perform infections at 37°C under 5% CO2 for 8-16h
using 1-2 ml of a mixture containing I volume of viral
supernatant, 1 volume of the target cells culture
medium, and 4~tg/ml polybrene. Within 24-48 h, cells
can be infected sequentially with all of the different
harvests of the same virus.
- Remove the last viral supernatants and replenish
all four dishes with the target cell culture medium,
using complete medium X for primary human fibroblasts.
Return cells at 37°C under 5% CO2.
- Let cells divide once or twice over the next 24-
48 h to allow integration of the viral genomes.
- Select cells for viral integrations using
puromycin. The exact dose of puromycin to use should
be determined in a preliminary experiment, in which
uninfected cells are exposed to increasing doses of
puromycin (e.g., 0, 250, 500, 750, 1000ng/ml) for 7-10
days; use the lowest dose that kills all cells. For
primary human fibroblasts, 500-750ng/ml is a good
starting point. Add puromycin to sample A, B, and C.
Leave sample D puromycin free.
- Maintain cells in continuous log-phase growth.
Samples that reach confluence should be trypsinized
and expanded to a larger dish; do not discard any
excess cells yet. Replace puromycin-containing media
as needed or twice a week until selection is complete.
Selection is complete when all of the uninfected cells
of control sample C have died, after typically 7-10
days of selection.
- Expand cells until samples A and B have
reached the size of a 100-mm dish. Count total number
of cells in samples A, B, and D. Evaluate the
number of population doublings done during the
infection/selection phases of the experiment (ΔPDi/s).
For this purpose, the simplest approximation is to
assume that samples A, B, and D have done an equivalent
number of doublings during this same period of
time. Using sample D as a reference, calculate ΔPDi/s knowing that ΔPDi/s = log[(final number of cells in
sample D) ÷ (initial number of cells plated in step 3)]
/ log. Discard sample D and set PD of samples A
and B to PDtime0 = PDi + ΔPDi/s.
- Cells are now ready to be tested for telomerase
activity and assessed for life span extension. Put
aside 5 × 104 cells of each sample for a telomerase
assay (see Section III,C). Start a growth curve by
seeding 2 × 105 cells of each sample in a 100-mm dish
(see Section III,C). Freeze remaining cells and label
vial with name of strain, vector transduced, population
doubling (PDtime0), and approximate number of
: Mix 10ml of DMSO with 90ml of
fetal bovine serum. Store aliquots at-20°C and keep
working solution at 4°C.
2. Measuring Cellular life Span
- Trypsinize cells as described earlier, resuspend in
culture medium, and recover by low-speed centrifugation
(2000g for 5 min at room temperature).
- Remove supernatant by aspiration, and resuspend
pellet in freeze medium so that each milliliter contains
the cell equivalent of 25-150cm2 of confluent
- Aliquot suspension in 2-ml cryogenic vials, at 1 ml
- Label vials with name of sample, population doublings,
date, and approximate number of cells.
- Place vials into a Nalgene Cryo 1°C freezing
container filled with fresh isopropanol. Transfer
- On the next day, transfer the frozen vials to either a
liquid nitrogen tank or a -135°C freezer.
To verify that exogenous telomerase has an
extended cellular life span, a growth curve is generated
to measure and compare the life span of the
vector- and hTERT-transduced cells.
3. Measuring the Activity of Telomerase Using
the Telomere Repeat Amplification Protocol
- Maintain the vector- and hTERT-transduced cells
in continuous log-phase growth as cells are counted
once a week. For this purpose, seed cells at a density
that requires a week for early passage cells to reach
confluence. For most primary human fibroblasts, a
density of 2 × 105 cells per 100-mm dish is adequate.
Take note of the population doublings of the two
samples, using the value of PDtime0 for cells that have
just been transduced and then selected.
- Let cells grow for a week at 37°C under 5% CO2.
- Trypsinize, wash, recover in culture media, and
then count cells using either a Coulter counter or a
- Calculate the number of population doublings
done by the cells since they were last seeded (or ΔPD),
where ΔPD = log[(number of cells recovered) ÷ (number of cell seeded)] / log.
- Calculate the current PD of each of the two cell
populations by adding the value of their ΔPD to that
of their previous PD (at which cells were when they
had been plated a week earlier). If ΔPD is negative, set
the value of ΔPD to zero.
- Replate cells at the exact same density as in step
I and freeze all remaining cells. Label frozen vials with
name of strain, vector transduced, population doubling
(current PD), and approximate number of cells.
- Repeat steps 2 though 6 until all of the vectortransduced
cells have become senescent. For both
samples, each cycle of growth will yield a ΔPD that is
then added to the previous PD to increase the current
PD to its present value.
- For each sample, plot PDn+1 as a function of time.
Cells transduced with the empty vector should eventually
reach a plateau corresponding to telomerecontrolled
senescence, at which point cells will cease
to divide. Pursue the experiment until the vectortransduced
cells have reached senescence. Cells can
be considered senescent once they perform less than
a doubling over a 2-week period. Most strains of
primary human cells reach senescence after 15-90 doublings.
A complete bypass of senescence by the hTERTtransduced
cells would then confirm that the cellular
life span has been extended by exogenous telomerase.
In this eventuality, grow the hTERT-transduced cells
until the life span has been extended by a factor of at
least 3, at which point the cells can be considered functionally
The TRAP assay is based on an improved version of
the original method described by Kim et al.
is typically performed using a commercial kit, the
TRAPeze telomerase detection kit (Serologicals Corp.,
- 50 mg/ml BSA: Dissolve 0.5 g of BSA in 10 ml of
deionized water. Aliquot and store at-20°C.
- 0.5M EDTA: To make 500ml, add 93.1 g of EDTA
(disodium salt) to 400ml of deionized water. Adjust
pH to 8.0 using sodium hydroxyde. Complete to
500ml with deionized water.
- 6X loading dye: To make 10ml, combine 6.4ml of
deionized water, 3 ml of glycerol, 600 µl of 0.5 M EDTA,
25 mg of bromphenol blue, and 25 mg of xylene cyanol.
- 12.5% acrylamide gel solution: To make 50ml,
combine 15.6ml of a 40% acrylamide:bisacrylamide
[19:1] solution with 5 ml of 5X TBE. Complete to 50 ml with deionized water. Just before casting the gel, add
50µl of TEMED and 500µl of 10% ammonium persulfate.
Mix well and cast immediately.
- 5X TBE: To make 1 liter, add 54g of Tris base,
27.5 g of boric acid, and 20ml of 0.5M EDTA, pH 8.0,
to 800ml of deionized water. Mix until solute has
dissolved. If needed, readjust pH to 8.1-8.5. Complete
to 1 liter with deionized water.
|FIGURE 1 Telomerase activity
in hTERT-transduced fibroblasts.
GM01604 fibroblasts engineered
to express exogenous hTERT or
hTERT were assayed for
telomerase activity using the
kit. H1299 and lysis buffer,
as positive and negative controls.
ITAS, internal TRAP assay
- Prepare the following samples for analysis:
uninfected cells, hTERT-transduced cells, vectortransduced
cells, and a positive control (telomerasepositive
cancer cell line, such as 293, H1299, or HeLa
cells). Trypsinize and count cells; for each sample,
transfer 5 × 104 cells to an Eppendorf tube.
- Pelet cells by low-speed centrifugation (2000g for
5min) and remove supernatant. Spin sample once
more for just a few seconds so that the very last drop
of remaining medium can be removed with the use of
a micropipette. Freeze pellets at -80°C.
- Thaw cell pellets on ice. To each sample, add
100µl of ice-cold TRAPeze kit CHAPS lysis buffer
(10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA,
0.1 mM benzamidine, 5 mM β-mercaptoethanol, 0.5%
CHAPS, 10% glycerol). Disperse cells by forcing the
pellets 5-10 times through the tip of a P200.
- Incubate cell lysates on ice for 30min.
- Spin samples in a microcentrifuge at 12,000g for
20min at 4°C.
- Transfer 80µl of the supernatant into a fresh
Eppendorf tube. Cell extracts should now contain 500
cell equivalents per microliter. Keep on ice and use
within the hour or else freeze and store at -80°C.
- End label the Telomerase Substrate (TS) primer
(5'-AATCCGTCGAGCAGAGTT-3'). To prepare a
sufficient amount of TS primer for six telomerase
reactions, combine 6µl of TS primer, 1.2µl of 10X
kinase buffer (provided with the enzyme), 3µl water,
1.5µl of [γ-32P]ATP (3000Ci/mmol, 10mCi/ml),
and 0.3µl of T4 polynucleotide kinase (10 units/µ).
Incubate at 37°C for 20min. Kill the enzyme at 85°C for 5 min.
- Prepare a master mix containing all components
of the telomerase assay, minus the extracts to be tested.
To prepare sufficient master mix for six assays,
combine 30µl of 10X TRAP reaction buffer (200mM Tris-HCl, pH 8.3, 15mM MgCl2, 630mM KCl, 0.5%
Tween-20, 10mM EGTA), 3µl of 50mg/ml BSA, 6µl of
dNTP mix (2.5mM each dATP, dTTP, dCTP, and
dGTP), 6µl of TRAP primer mix (contains the PCR
primers needed for amplifying the telomerase products),
234.6µl of water, 12µl of 32P-labeled TS primer (from the previous step), and 2.4µl of Taq DNA polymerase
(5 units/µl). Mix well. Aliquot the master mix
in four RNase-free PCR tubes at 49µl/tube.
- On ice, thaw all of the cell extracts to assay.
Samples should include the uninfected cells, vectortransduced
cells, hTERT-transduced cells, a negative
control (TRAPeze kit 1X CHAPS lysis buffer), and a
positive control (telomerase-positive cancer cell line).
- Add 1 µl of sample per tube (500 cell equivalents),
mix, and place in the thermocycler.
- Incubate at 30°C for 30min.
- Perform the following two-step PCR for 27-30
cycles: 94°C for 30s, followed by 59°C for 30s. While
these conditions work on most thermocyclers,
optimization of the annealing temperature and addition
of a 72°C extension step may be necessary on
- Add 10µl of 6X loading dye to all samples. Store
at -20°C in a β blocker.
- Prepare a vertical 12.5% polyacrylamide gel.
Choose glass plates, spacers, and comb so that the gel
will be 1.5mm in thickness and 10-12cm in length.
Once the mold is ready, add TEMED and ammonium
persulfate to the 12.5% acrylamide gel solution and
pour the gel immediately.
- Once the gel has polymerized, load 30 µl of each
sample per lane. Run at 400V for 90min or until the
xylene cyanol has run 70-75% of the gel length.
- Dispose of the electrophoresis buffer as liquid
radioactive waste. Wrap gel in saran wrap.
- Expose gel to an X-ray film or PhosphoImager
- The presence of an active telomerase should
yield a ladder of products with 6-bp increments, starting
at 50 nucleotides. For the TRAP assay to be valid,
the following conditions should first be met: (1) the
negative control corresponding to the lysis buffer
should display no such ladder; (2) the positive control
(e.g., 293, H1299, or HeLa cells) should yield an intense
ladder; and (3) all samples should display a 36-bp
band corresponding to the internal TRAP assay standard
(ITAS), a control template included in the TRAP
primer mix whose amplification serves to document
the efficiency the PCR step of the protocol. The lack of
ITAS amplification would suggest that inhibitors of Taq DNA polymerase may have been present in some of
the samples. If the assay has met these conditions,
analysis of the experimental samples can now proceed.
A successful reconstitution of telomerase activity by
exogenous hTERT should produce a telomerase ladder
similar in intensity to that of the positive control, with
no such activity detected in either vector-transduced
cells or uninfected cells.
D. Example of the Use of hTERT to Extend
Cellular Life Span
1. Primary Human Fibroblasts
|FIGURE 2 Extension of cellular life span by exogenous
GM01604 fibroblasts engineered to express
exogenous hTERT or no
hTERT were maintained in
continuous log-phase growth and
counted once a week.
Population doublings executed expressed as
a function of time.
Primary human fibroblasts (GM01604; Coriell Institute,
Camden, NJ) were infected with pBabePurohTERT
retroviruses under the conditions described in
this article. Infected cells were selected for 2 weeks
with 750ng/ml puromycin and were then tested for
telomerase activity using the TRAPeze telomerase
detection kit. Figure 1 shows a strong telomerase
ladder in both hTERT-transduced cells and positive
control H1299. The 36-bp ITAS is visible in all lanes,
indicating that inhibitors of Taq
which could have resulted in false negatives, were
absent. The life span of hTERT-transduced cells was
then compared to that of uninfected cells. Cells were
maintained in continuous log-phase growth and
counted once a week. Figure 2 is a graphic representation
of the cumulative number of PD executed by the
cells as a function of time. Note that hTERT minus cells
ceased dividing at PD 60 whereas hTERT-transduced
cells grew beyond PD 180, at which point these cells
were considered functionally immortal.
- Other retroviral vectors carrying hTERT are
available from Dr. Robert A. Weinberg. (Whitehead
Institute for Biomedical Research, Cambridge, MA).
These additional vectors include an alternate version
of vector pBabePuro-hTERT, as well as plasmid
pCIneo-hTERT, a retroviral construct that confers
resistance to G418. Be sure to use a vector carrying an
hTERT cDNA that has not been epitope tagged at the
C terminus, as these modifications can block the access
of telomerase to the telomeres (Ouellette et al., 1999).
- To adapt the protocol described for other
primary human cells, replace complete medium X
with a culture medium that is compatible with the
long-term growth and survival of your target cells.
Transfected ΦNX-A cells can be made to produce
viruses in a large variety of culture media.
- It should be noted that hTERT alone may not be
sufficient to immortalize all types of primary human
cells. First, the enzyme telomerase does not appear to
alter the phenotypic properties, such that postmitotic
terminally differentiated cells are unlikely to be
rescued by the enzyme. Second, certain primary human cells experience additional forms of senescence
that are independent of telomere size (Kiyono et al.,
1998). It has been suggested that these extra barriers
represent a stress response to inadequate culture
conditions, in some cases elicited by the loss of mesenchymal-
epithelial interactions (Ramirez et al., 2001;
Shay and Wright, 2001). In addition to exogenous
hTERT, the immortalization of such primary cells
might also require a reoptimization of the culture
conditions for long-term growth, the cultivation of the
cells over feeder layers, or, alternatively, the use of
oncogenes that can block pRB function, such as the
SV40 large T antigen, HPV type 16 E7, or adenovirus
type 5 EIA.
- When working with amphotropic retroviruses,
due caution must be exercised in the production, use,
and storage of recombinant viruses. Transfected ΦNXA
cells, viral supernatants, and all plasticwares that
have been in contact with these reagents should be
bleached and treated as biohazard.
- When using 32P, appropriate measures must
be taken to ensure that the user remains shielded
from radiation and that radioactive by-products and
wastes are being contained appropriately. Working
areas should also be monitored for radioactive
- When performing the TRAP assay, precautions
should be taken to limit PCR contamination. To limit
such contamination, electrophoresis analysis of the
samples should be run in a separate area away from
the bench where samples are prepared and TRAP reactions
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