At UC Berkeley Gene targeting facility, we offer technical advice and assistance to investigators with in vivo manipulation of the mouse embryo. The facility is accredited by AAALAC International and is pathogen free.
The following procedures are routine:
- Pronuclear DNA injection- transgenic mouse
- Blastocyst ES cell injection-germ line chimeras
- ES cell culture and gene targeting
- Mouse sperm cryopreservation
Transgenic mouse
For pronuclear injection, we will use F1 zygotes of CBA X C57BL6 strains or C57BL6 if absolutely necessary. Investigator should supply purified DNA accompanied by required records. Only constructs that meet our quality control requirements will be injected.
After potential founders are weaned, the investigator will perform genotyping from the tail tissue that we provide. A minimum of three transgenic founders will be generated per given construct.
Germ line chimeras
Investigator will provide desired clones of the ES cells accompanied by the records specified in the ES cell injection form . We will use C57BL6 or BALB/c host blastocyst, depending on the background of the targeted line. Chimeras expressing the coat color of the targeted line will be generated for investigator to proceed with screening. To establish the mutant mouse strain progeny, the investigator will further breed the altered fertile male mice. The germ line transmission is highly depended on preserved capacity of ES cell line. Thus, good cell culture practice is of critical importance.
Es Cell culture and gene targeting
Investigators may elect to have their gene targeting experiments handled by us. In that case, we will culture the parental ES cell line, perform electroporation, antibiotic selection, individual colonies picking, replica plating and the short term freezing (while genotyping is being completed).
Typically we would use TC-1 parental line although other ES lines of 129 SV background as well as C57BL6 background are available. Investigator should provide isogenic, linearized, stringently purified* DNA fragment. We require 4 micro grams of DNA per Kb of the vector construct resuspended in sterile water.
Aproximately three weeks after accepting the DNA we will hand out the set of replica plates to the investigator for screening. When the appropriate colonies are identified, we will thaw them and provide the samples for further confirmation and injection into blastocyst.
Sperm cryopreservation
An economical way to preserve mutant mouse line is sperm cryopreservation. For this purpose, investigator provides one or two fertile mice (4 month old) of desired line. One week after mating with wild type female mouse, the male mouse is sacrificed for sperm collection. Collected sperm will be cryopreserved in the two different LN2 tanks or in the investigators lab.
If necessary, we can check the viability of the frozen sperm by thawing and performing in vitro fertilization.
In the past we showed that 50% of the eggs can be fertilized by the thawed sperm and develop into 2 cell stage embryos. Approximately 10% of the transferred fertilized embryos develop to full term.
The following are links to download forms in PDF format. To view them,
download the free Acrobat Reader from Adobe.
Protocol for Preparation of DNA for Transgenic Mice
