How to Make Stable Transfected Cell Line

Ensure that only one cell is present per well after the transfer.

1. Step 1 : Transfect cells. Transfect the cells using the desired transfection method. ...
2. Step 2 : Passage cells with antibiotic. ...
3. Step 3 : Monitor for cell "islands" ...
4. Step 4 : Isolate colonies. ...
5. Step 5 : Transfer single cells.

1. How do you make a cell line stable?
2. How do you choose colonies for stable cell lines?
3. What are stable cell lines?
4. How do you create a cell line?
5. Where are stable cells found?
6. How do you select a transfected cell?
7. What is the meaning of cell lines?
8. What is the difference between stable and transient transfection?
9. How do you use a clone cylinder?
10. What is stable gene expression?
11. Can stable cells regenerate?
12. What is a permanent cell?

How do you make a cell line stable?

The protocol for generating stable cell lines requires several steps as shown below:

1. Generate a kill curve to determine the optimal selection antibiotic concentration.
2. Transfect cells with desired plasmid construct(s)
3. Select and expand stable polyclonal colonies.
4. Identify single clones by limited dilution and expansion.

How do you choose colonies for stable cell lines?

To pick colonies, prepare 24-well dishes with 1 ml media containing G418 in each well. Rinse the dish with PBS and then add warm PBS containing 5% trypsin (1 ml standard trypsin-EDTA plus 19 ml PBS).

What are stable cell lines?

Generation of a stable cell line refers to the process of developing homogenous populations of cells that demonstrate expression of a transfected gene insert. ... This is opposite of transient transfected cells that express the transfected DNA for a short time (e.g. 8 to 96 hours).

How do you create a cell line?

The simplest way to create a new cell line is to modify an existing one, a common strategy when an established line already comes close to meeting the requirements. Cells optimized to grow particular viruses or maximize recombinant protein production often come from such modifications.

Where are stable cells found?

In cellular biology, stable cells are cells that multiply only when needed. They spend most of the time in the quiescent G₀ phase of the cell cycle but can be stimulated to enter the cell cycle when needed. Examples include the liver, the proximal tubules of the kidney and endocrine glands.

How do you select a transfected cell?

One of the most reliable ways to select cells that stably express transfected DNA is to include a selectable marker on the DNA construct used for transfection or on a separate vector that is co-transfected into the cell, and then apply the appropriate selective pressure to the cells after a short recovery period.

What is the meaning of cell lines?

Cell line is a general term that applies to a defined population of cells that can be maintained in culture for an extended period of time, retaining stability of certain phenotypes and functions. Cell lines are usually clonal, meaning that the entire population originated from a single common ancestor cell.

What is the difference between stable and transient transfection?

In stable transfection, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell. However, in transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome.

How do you use a clone cylinder?

Using sterile medium forceps, pick up a cloning cylinder. Gently press the flat bottom of the cylinder into the smooth silicone grease and remove with a sudden vertical motion. If done properly, this will give even distribution of grease on the bottom of the cylinder. Set the cylinder over a colony.

What is stable gene expression?

Expression of the foreign genetic code is continual and the transfection procedure is termed a stable transfection. If the host cell rejects the inserted DNA segment, the transfection becomes transient because expression of the introduced genetic material is lost with subsequent generations.

Can stable cells regenerate?

Labile cells (1) such as squamous epithelium of the skin continuously divide and regenerate. Permanent cells (2) such as neurons are unable to proliferate and leave the cell cycle. Stable cells (3) such as liver hepatocytes have a low level of replication and can regenerate when signaled.

What is a permanent cell?

Permanent cells are defined as cells that are unable to replicate in postnatal life. Nervous cells, also termed neurons, together with skeletal muscle and cardiac cells, are included in this group, which traditionally identifies the human tissues that are incapable of spontaneous regeneration.