somatic cell hybridization assay
somatic cell hybridization assay
With the advent of molecular genetics techniques, important advances have been made in somatic cell hybridization: the DNA used as a probe can be obtained from the cells used for somatic cell hybridization or Southernblot, and probes from the corresponding genes can be identified whether they are hybridized to a filter membrane or to a denatured or non-denatured fragment of a chromosome. However, due to the similarities between humans and rodents at the DNA and protein level, the fragments used as hybridization must be very specific to avoid cross-reactivity with rodents. Many genes have been successfully localized using this technique, but it is worth noting that due to the presence of pseudogenes and gene families, probes should be designed to match as closely as possible to the corresponding fragments of the gene to avoid cross-reactivity with these genes.
Operation method
Monolayer whole cell fusion assay
Materials and Instruments
Recipient cells that have reached a certain level of confluence Move Basic scheme Single layer whole cell fusion 1. If the cell selection conditions are not known, determine them by passing recipient cells cultured in 25 cm culture flasks at a ratio of 10:1 into different culture flasks with different levels of selection reagents. Cells were observed twice a week, fluids were changed, and after 10 d the lowest concentration of the reagent that inhibited cell growth was determined. 2. In the afternoon of the day before the fusion, equal numbers of donor and recipient cells (usually 5X105?IXlO6 cells) are cultured in IOml of medium with serum, while donor and recipient cells are cultured separately in medium with or without PEG for control, and incubated overnight. Donor and recipient cells should die under selection conditions, PEG-containing cells should recover and begin to confluence under selection conditions, and when a new cell line or a new PEG is used, the latter control should be repeated. 3. Observe the cells under the microscope, they need to be 70% confluent, and the cells need to be in good contact with each other, but not squeezing each other, if the amount of cells is not enough, prepare new flasks to increase the culture. 4. Aspirate the medium out of the bottle, wash it twice with serum-free medium and aspirate it completely, tilt the bottle for a while and aspirate the last of the medium. 5. Add 2 ml of 50% PEG solution at 37°C. Tilt the bottle back and forth to mix the solution quickly in the cells and let it stand for 2 min (time is very important). If necessary, optimize the conditions by the following tests: peg concentration of 44%~50%; incubation of monolayer of cells for 2~3 min, suspension of cells for 2~4 min. 6. Gently add 10 ml of serum-free medium from one side of the dish, gently invert it to make PEG diffuse completely, aspirate the medium and discard it, repeat the washing with serum-free medium twice, and the last time with 10 ml of serum medium, because the cell membrane is very fragile at this time, so the above operations need to be done gently. 7. Add 10 ml of serum medium and incubate for 36~48 h (the cells will be expanded twice during this period). 8. Digest the cells with trypsin and divide them into 5-10 10 cm plates. To ensure selection, the concentration of cells in the plates needs to be low to ensure that the cells can divide several times before confluence. Add 10 ml of serum medium and the appropriate concentration of selective reagent to each plate (step 1) and incubate at 37°C. 9. Change the solution every 5d until clones have grown, and check for clones in the dish 10?14d after changing the selective medium. Avoid damaging the cells or making them grow larger, as moving the cells may cause them to grow sister clones. 10. Pick out the clone with a clone picking needle (Support Option 1). 11. To avoid maintaining and expanding unwanted cell lines, expand, characterize (steps 3-5), and freeze the clones as soon as they are grown (Appendix 31). To avoid chromosomal deletions and recombination, passaging should be minimal (one freezing tube for up to three generations of cells, two freezing tubes for a 6 cm dish). Multiple identification methods are used to characterize the cell line, and re-identification is required during recovery and expansion to ensure that there is no recombination or segregation of chromosomes. If necessary, subclone cell lines that have isolated additional chromosomes (Supporting Option 2) 12. For rapid recovery, culture the cells into 25 cm tissue culture flasks and increase the serum content of the freezing medium if the cells recover slowly after freezing. 1. Digest adherently grown recipient cells, resuspend in medium, count donor and recipient cells using a blood cell counter, take 2XIO 6 each and mix in 15 ml snap-cap polypropylene tubes, and maintain to ensure valid selection. 2. Centrifuge at 400 g for 5 min at room temperature, add IO ml of serum-free medium to the recipient cells, centrifuge again and carefully aspirate the medium, tilting the tube to absorb the medium. 3. Flick the tube to loosen the mass. Pipette 0.3 ml of 50% PEG solution at 37°C and add along the wall of the tube while shaking or flicking to disperse the mixture. Incubate at room temperature for 3 min. If necessary, optimize the fusion conditions (basic protocol, step 5). 4. Add 8 ml serum-free medium and centrifuge immediately at 400 g for 5 min. 5. Carefully aspirate the medium and resuspend the cells in IOml serum-free medium. Centrifuge again. At room temperature, aspirate the medium and resuspend the cells in IOml serum medium. Be extremely careful during the washing process, as the membranes of the cells are fragile at this point. 6. Add 3 ml of cell suspension to each IOOmm tissue culture plate. Add ImI cell suspension to another 100 mm plate as control. Incubate for 36-48 h. 7. Incubate the cells in medium with serum and screening factors (without screening factors as control). 8. Prepare the clone and identify it (basic protocol, steps 9-12). For more product details, please visit Aladdin Scientific website.
Medium suitable for recipient cells Suitable selective reagents Donor cells with chromosome of interest and suitable genetic markers
10 cm tissue culture plates 25 cm tissue culture flasks Inverted phase contrast microscope
