Experimental procedure for luciferase gene labeling of tumor cells
In mammalian bioluminescence, the Firefly luciferase gene (constituted by 554 amino acids, about 50KD), that is, the luciferase gene, is integrated into the chromosomal DNA of the cell to be observed to express luciferase, and the luciferase can be stably expressed. In cell lines, luciferase is also stably expressed when cells divide, metastasize, and differentiate. After the labeled cells are inoculated into the experimental animal, luminescence can be produced in a few minutes when exogenously (either intraperitoneally or intravenously) is administered to the substrate luciferin. This enzyme catalyzes the oxidation reaction of fluorescein in the presence of ATP and oxygen, so that luminescence is only produced in living cells, and the intensity of luminescence is linearly related to the number of labeled cells.
Using the lentiviral expression system GFP (mCherry) and Luciferase2 double-labeled expression cell line, GFP (mCherry) and Luciferase2 were ligated by 2A sequence, and GFP (mCherry) and Luciferase2 were simultaneously expressed in vitro, which facilitated in vitro observation and in vivo tracing; mCherry was available. Traced in vivo, combined with a new generation of Luciferase2, can easily monitor the growth and changes of cells in the body.
Description of the transfection method and labeling process (Lentivirus transfection Hygromycin screening):
(a) plasmid part
1. Digestion vector: pGL4.10 (luc2) was digested to obtain a 1400 bp luc2 gene fragment; EGFP and mCherry fragments were amplified by PCR to obtain a fragment of about 700-800 bp; pSin-hyg-T2A vector was linearized.
2. Electrophoresis and gel recovery: The above-mentioned enzyme-cut product DNA electrophoresis, TAKARA kit gel recovery, and the recovered product was identified by a small amount of electrophoresis.
3. Ligation vector and the fragment of interest: The luc2, EGFP or mCherry fragment and the pSin-hyg-T2A linearization vector were ligated using Invitrogen T4 ligase, and ligated using a 20 ul system at 25 ° C for 10 min.
4. Transformation: After the One Shot Stbl3 was melted on ice, the ligation product was added, and the mixture was allowed to stand for 25 min, then heat-shocked in a water bath at 42 ° C for 45 sec, and then 250 ul of anti-culture medium 225 shakes were added for 1 h to transform the bacteria. Place on an ampicillin-resistant plate at 37 degrees overnight.
5. Pick the monoclonal: After observing the plate, pick 15 monoclonal clones into the shaker tube containing 2 ml of ampicillin-resistant LB medium, and shake the bacteria for 6.5 hours.
6. Small plasmid and identification: The plasmid was lysed with 25 ul of RNase containing ddH2O; the recombinant plasmid was identified by restriction enzyme digestion.
7. Identification of fluorescein expression: Recombinant plasmid pSin-hyg-GFP/luc2 (hereinafter referred to as Gluc) or pSin-hyg-mCherry/luc2 (hereinafter referred to as Mluc) was transfected into 293T cells: after quantification of DNA, transfection with Gluc or Mluc using PEI To 24-well plates pre-plated 293T cells. Transfection was transfected by liposome method using JetPEI (Polyplus). Add 50 ul of normal saline to each of the two tubes of EP tube, one tube was added with 1 ug of plasmid (DNA volume = 1 ug / DNA concentration), gently shake and mix, then gently centrifuge; add PEI 2ul in another tube and gently shake Mix well, centrifuge gently, then add the solution in the PEI tube to the containing plasmid tube and incubate for 20 min at room temperature. The liposome-encapsulated DNA mixture was added to 24 wells to be transfected, cultured at 37 ° C, 5% CO 2 , and the culture medium was changed after 8 hours.
8. Reporter gene detection: Passive Lysis Agent lysed cells, added luciferase substrate, and transfected plasmid cells with negative and positive controls for single reporter gene detection.
9. Extraction in the plasmid: Take 1 ml of the transfected reporter gene and test the correct bacterial solution. Add 100 ml of ampicillin-resistant LB medium to shake the bacteria overnight, use the Invitrogen kit to extract, product electrophoresis, and store at -20 °C.
(2) Lentiviral packaging part
1. Plasmid co-transfected cells: One day before transfection, the cells of 293T in a 10 cm culture plate reached 90%, and after trypsinization, 1:3 was introduced into a new 10 cm culture plate. At the time of transfection, the cells reached a density of 80% in the culture plate. The plasmid VSV G, Packaging Mix encoding the lentiviral envelope protein and 15 μg of the constructed Gluc or Mluc plasmid were co-transfected by liposome PEI transfection. Fresh DMEM medium was changed after 6 h.
2. Extraction of virus supernatant: After transfection for 48-72 hours, the medium containing the virus solution was transferred to a 15 ml centrifuge tube, and the pellets of the pellet were removed by centrifugation at 3000 rpm for 10 min at a low temperature of 4 °C.
3. Concentrated virus: The virus supernatant was filtered through a 0.45 um filter into a concentrated centrifuge tube, centrifuged at 2000 rpm, 4 ° C, and 15 min. The concentrate was collected in a 500 μl centrifuge tube and stored at -80 °C.
(iii) Lentivirus infection & screening
1. On the day before infection (Day 1), the cells of interest were digested and counted, the cells were plated in 24-well plates (to achieve a confluence of 30%-50% before infection), and the cells were incubated overnight at 37 °C.
2. (Day 2) The cryopreserved lentiviral solution was thawed, and 200 μl of a 1:2 virus dilution was prepared and added to the cells.
3. Add Polybrene to each well to a final concentration of 6 ug/ml, shake well and incubate overnight at 37 degrees.
4. (Day 3) Remove the medium containing the lentivirus and add 500 μl of complete medium.
5. (Day 4) Change the medium and add 300 μg/ml Hygromycin (Sigma) to select stable infected cell clones.
6. (Day 4-7), change the solution every 24 h, 300 μg/ml Hygromycin (Sigma) to select stable infected cell clones.
Maintain culture.
7. (Day 8-12) Cells were gradually cultured from 24-well plates into 6-well plates, 6 cm plates, and 100 μg/ml Hygromycin in 10 cm plates.
8. Target cell-Gluc/Mluc cell line identification: Take 5×104 cells, lyse with Passive Lysis lysate, add luciferase substrate, and transfect the plasmid cells with negative and positive control for single reporter gene detection. . GFP and mCherry expression were observed by fluorescence microscopy.
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