Telomerase plays an extremely important role in the stability of chromosomes and in determining the cellular life cycle, and is mainly used in (1) the development of transgenic technology (2) gene-induced expression.
Operation method
Immortalization of human mesenchymal stem cells induced by telomerase
Principle
1. Cells extracted from animal or human tissues will divide and proliferate to different degrees in in vitro culture, which is called proliferative senescence. However, some cells can break through proliferative senescence and have the ability to proliferate indefinitely when induced by spontaneous or other conditions, and become immortalized cells. bone marrow-derived mesenchymal stem cells are pluripotent stem cells, which can be used as a source of seed cells for tissue engineering. 2. Telomerase plays an extremely important role in the stability of chromosomes and in determining the life cycle of cells. Telomerase or terminal transferase is composed of two main subunits: the RNA component (hTR) and the proteolytic subunit (hTERT). The RNA (hTR) subunit is universally expressed in normal and tumor tissues, whereas the proteolytic subunit (hTERT) is expressed only in tumor cells, germline cells, and activated lymphocytes. 3. The mechanism of cellular senescence is now widely accepted. The two-phase model of cellular senescence, the M1/M2 model, is now widely accepted. During cell growth, after many divisions, the cell's response to growth factors disappears under the action of tumor suppressors, such as p53 and pRb, with the disappearance of the mitotic response and the production of DNA synthesis inhibitory proteins, DNA synthesis stops, the cell irreversibly stagnates in the G1 phase, and senescence occurs (sessence. M), and apoptosis or cell death occurs. The cell undergoes apoptosis or death. immortalization refers to the process by which cells cultured in vitro escape from proliferative senescence, either spontaneously or by external influences, and thus have the ability to proliferate indefinitely.
Materials and Instruments
Human mesenchymal stem cells Move Material Growth medium: Dulbecco's modifiled Eagle's medium (DMEM) with high glucose concentration (4.5 g/L), L-glutamine 2 mmol/L, 10% fetal bovine serum, 100 U/mL penicillin, and 100 ug/mL streptomycin. Polyglutamine: 8 mg/mL HEPES 0.5 mol/L, pH 7.1 200uL NaCl (5 mol/L) 600 uL PCR reagents PD in the formula represents the number of population doublings, In is the natural logarithm, Nstart is the initial inoculum of cells, and Nfinish is the total number of cells obtained during the passaging culture. In Example 2, if 2x105 cells are seeded initially, the number of cells can be increased to 3. 2x106 after culture. If the cells are cultured 10 times with a 1:4 cell site, then: Justice primer 2uL Caveat A reservoir of transduced cells, which are at different levels of population multiplication (PI) (which can be matched to the PD growth curve), is created by dispensing the cells into ampoules for freezing and preservation. This also saves time because if contamination occurs the cultured cells have to be discarded and started over.After the immortalized cell line has been established, it must be checked for authenticity to ensure that it originated from the original material and was not derived from other immortalized cell lines in the laboratory due to cross-contamination. Common Problems Immortalization of human bone marrow mesenchymal stem cells by reference transduction of the human telomerase reverse transcriptase gene For more product details, please visit Aladdin Scientific website.
Glucose L-glutamine Fetal bovine serum Penicillin Streptomycin Polyglutamine
Culture Flasks Porous Plates
non-sterile
Plain container: culture bottle 25 cm2, 75 cm2
Materials for the production of reverse transcriptase viral vectors
Cell line: PG13
PG13
GP+E-86
Retroviral vector GCsam hTERT
htrt DNA for transfection
Tx Buffer: 20 mL total, containing the following:
NaCl, 5rnol/L 100uL
Na2 HPO4/NaH2PO4, 1 mol/L 3uL
UPW 1.7 mL
Buffer A: 20.04 mL total, containing the following:
EDTA (0.5 mol/L) 40uL
Tris-HCl, pH 7.5 (0.5 mol/L) 400 uL
UPW 19 mL
Materials for transduction of hMSC (human mesenchymal stem cell) cells
hMSC (human mesenchymal stem cell) cells
Culture flask 75 cm2
Multiwell plate 6 wells
Post-transduction materials
laboratory material for cryopreservation (see protocol 20.1)
laboratory material for DNA fingerprinting (see Scheme 16.8) or DNA profiling (see Scheme 16.9) or other validated analytical methods (e.g. Scheme 16.10)
DNA 100 ug/mL
10x PCR buffer L (containing Mg2+ )
Forward primer (2umol/L)
Antisense primer (2umol/L)
dNTP mixture (10 mmol/L)
DNA polymerase
UPW
Procedure
Generation of Reverse Transcriptase Viral Vector
Reverse transcriptase viral vectors with the hTERT gene were packaged into the gibbon ape leukemia virus (GALV)-packaged cell line PG13 in a two-step process.First, the packaged cell line GP+E-86 was transfected using 20 ug/mL htrtDNA (Geron), and then the PG13 cells were infected with the supernatant.
1. 6.7x105 GP+E-86 cell line was inoculated in a small culture flask (25 cm2 ).
2. Transfection of GP+E-86 cell line
(a) Add 280uL of Tx buffer to each tube (common container).
(b) Prepare DNA tubes:
Composition Name DNA Concentration 15 ug DNA Buffer A CaCl2 2 .5mol/L Total amount
undefinedug/uL undefineduL 280-30-X 30 280
~undefined X x Z = 15 ug
(c) Add the Tx buffer drop by drop to the tube containing the DNA solution and mix gently.
(d) Incubate at room temperature for 30°C to produce a precipitate.
(e) To each 25 cm2 culture flask containing GP-E-86 cells, add 5 mL of fresh culture solution.
(f) Gently add the mixture (Tx+DNA) to the GP+E-86 cells and incubate for 4-6 h at 37℃.
(g) In a 25 cm2 culture flask, carefully wash three times with D-PBSA.
(h) Add fresh culture medium to the cells and incubate at 37°C overnight. 3. Replace the cell culture medium, add fresh culture medium to the cells and incubate at 37°C overnight.
3. Replace the cell culture with 2 mL of fresh culture medium (replacing the previous 5 mL of culture medium) to produce virus.
4. Inoculate 1x104 PG13 cells into each well of a 6-well plate on the same day as step 3.
5. The following day, supernatant from infected GP-E-86 cells is harvested and a final concentration of 8 ug/mL of polyamine (e.g., 1 ug/mL supernatant) is added.
6. Filter the supernatant through a 0.45 um pore size membrane and add 2 mL of filtrate to each well containing GP-E-86 cells.
7. Centrifuge the plate at 1000 g at 32 °C.
8. Incubate at 37°C overnight.
9. On the following day, change the culture medium of the cells.
Transduction of hMSC cells
1. 2x106 PG13 cells were seeded into 75 cm2 culture flasks.
2. On the following day, 6 mL of fresh culture medium was added to the PG13 cells.
3. On the third day, hMSC cells (about 2. 5x104~7. 5x104 cells) were added to 6-well plate for transduction.
4. The supernatant was harvested from the packed cells, and polyamine at a final concentration of 8 ug/mL was added, and the supernatant was filtered through a 0.45 um pore size filter membrane.
5. Add 2 mL of filtered retroviral supernatant to each well of a 6-well plate.
6. Incubate the plate at 1000 g at 32°C and incubate at 37°C overnight.
7. Aspirate off the retroviral supernatant and add fresh medium.
Handling of cultures after transduction
1. After 10-12 passages, cells that have not received the hTERT gene will die, and the remaining cells will be sure that the hTERT gene has been inserted, and cells with the hTERT gene will be selected during the passaging process.
2. It is recommended to freeze the cells in ampoules to create a reservoir of transduced cells that are at different levels of population increase (PI) (which can be matched to the PD growth curve). This also saves time because if contamination occurs the cultured cells have to be discarded and started over.
3. Obtain the PD during plum passaging culture using the following formula to establish the PD growth curve.
Example 1, if 2x105 cells were initially seeded and increased to 3. 2x106 cells after culture:
If a passaging culture is performed with a cell volume of 1:4, the Nfinish after each passaging culture will be multiplied by 4. So in the above example, assuming 10 passaging cultures, the Nfinish would be (3. 2x106 )x4 times 10, or 3. 4x1012.
4. After an immortalized cell line has been established, it must be checked for authenticity to ensure that it originated from the original material and was not derived from other immortalized cell lines in the laboratory due to cross-contamination (see Section 19.5). This can be done, e.g. by PCR testing for the transfer gene (hTERT) (see below), DNA fingerprinting (see Scheme 16.8) or DNA profiling (see Scheme 16.9).
PCR for hTERT
1. Dissolve PCR reagents and store on ice.
2. carefully mix the following reagents, remembering to include both a positive control (other hTERT-positive specimen) and a negative control (no DNA template)
DNA 1uL (100ng) 100 ug/mL
10xPCR buffer (with Mg2+ ) 2uL
Antisense primer 2uL
dNTP mixture (10 mmol/L) 0.4uL
DNA polymerase 0.1uL
UPW 12.5uL
Final volume 20 mL (including DNA)
3. Place the tube in the PCR instrument and proceed as follows: 90 ℃, 3 min, denaturation first; 94 ℃, 30 s, denaturation again; 59 ℃, 39 s, re-denaturation; 74 ℃, 1 min, extension; and so on for 30 cycles, followed by 74 ℃, 1 min.
4. The PCR products were electrophoresed on a 1.5%-2% agarose gel.