Investigators must receive approval from the director of the core for a project to be initiated.
Complete and submit the ROSA Targeting Request Form.
In order to initiate a project, investigators must submit the following information:
- A set of standard maps showing three things for each gene targeting event:
1. The genomic locus to be targeted;
2. The targeting vector construct;
3. The predicted structure of the targeted locus.
These maps should make clear the predicted wild type and targeted restriction patterns that you will see with both 5' and 3' probes. It is beneficial to have genomic Southerns of 129 or C57 DNA with the appropriate digests for both the 5' and 3' probes to ensure that they are clean and ready to go prior to scheduling an electroporation. You may contact the core's supervisor for a dry cell pellet in order to extract DNA for this purpose.
- A photograph of each digest must be provided to the core along with the purified DNA.
DNA must be digested, phenol/chloroform extracted and precipitated in EtOH. Please, refer to the Preparation of DNA for Electroporation Protocol for details on how to prepare DNA. The core will only accept DNA in precipitate form, in 100% EtOH, in a tube filled to capacity with 100% EtOH in order to minimize chances of contamination in the cell culture process. 60ug of linearized DNA must be submitted for each ROSA targeting construct to be electroporated.
Investigators must bring DNA to the core the Thursday prior to the date of electroporation.
Electroporations should not be initiated unless there is a clear and firm commitment to perform analysis on the clones immediately. It is highly recommended to have Mini Southern data available within three months of freezing 96 well plates. Cell recovery may not be optimal if plates are stored for long periods at -80°C.
In an effort to avoid confusion, submit a history of any electroporation in our core at a prior date if applicable.
It is the individual investigator's responsibility to run Mini Southerns.
Mouse embryonic stem cells will be electroporated using ROSA targeting constructs prepared by individual investigators and submitted to the core as described in section A above.
The core defines one ROSA target as a ROSA DNA construct electroporated into one cell line where a total of 24 colonies are picked for $500. Therefore a total of four different ROSA targeting constructs may be placed in one 96 well plate.
Colonies will be frozen in 96 well plate formats, at which time duplicate plates will be cultured for DNA analysis by investigators. Approximately one week after colonies are frozen core will notify investigators to pick up plates for Mini Southern analysis. Investigators will receive duplicate plates for each 96 well plate picked, in lysate form, in order to begin DNA precipitation for analysis. These plates should be used in probing 5' and 3' ends.
After mini southern data is received, the core will expand clones for investigators at an additional charge.
After contacting the director of the core for project approval, investigators must complete and submit the ROSA Targeting Request Form. The core supervisor will e-mail the contact person within three working days after the form is submitted to inform the lab of the scheduled electroporation date. Every request will be taken on a first come, first serve basis.
Colonies will be picked seven to ten days after electroporation.
Plates will be frozen three to four days after picking colonies.
Investigators will be notified to pick-up lysate plates one week after plates are frozen.
Therefore, investigators should be prepared to begin Mini Southern analysis of lysate plates approximately 17-19 days after the scheduled electroporation day.
In the event that the culture becomes contaminated, the core will immediately repeat the electroporation at no additional cost to investigators.
The director of the core will deal with other problems on a case by case basis.
After service agreement:
We would appreciate, that any publications arising from the generation of cell lines by this core carry an appropriate acknowledgment of this service.