Nevada Transgenic Center

Services

Pricing
Please contact the Department of Pharmacology at 775-784-6956 for pricing information

ES cell gene targeting
Electroporation: We will electroporate your targeting vector into 129 ES cells, select, and provide you with DNA from 100 clones of cells that we will store in our freezer. Upon analysis we will thaw and expand positive clones and provide you with at least 2 ampoules of 5x10⁶ cells each.

You should provide 40 μg of linearized, purified DNA in a total volume of less than 200 μl. There should be a minimum of 4 kb of homology with genomic DNA.

For Gene Targetting into R1 ES Cells:

Per electroporation (usually we do 2 simultaneous electroporations per construct):

We need:

1. 20-25μg linearized DNA in ~100ul, i.e. a total of 40-50μg DNA for 2 EP's
2. map of construct

We will try to pick as many clones as possible. Usually we can get at least 100 clones and may get as many as 400 clones/50μg DNA.

We will freeze one 96 well of each clone and make duplicate plates for DNA analysis. Each well contains ~4μg DNA.

From time of electroporation until time that DNA is isolated is 3 weeks.

If you are doing the DNA analysis, you should try to get results back within 2 months so we can thaw and refreeze positive clones and provide you with more DNA.

If we do the analysis you should provide us with PCR primers and PCR conditions.

Microinjection of blastocysts
We will inject your targeted ES cells into blastocysts of your choice. We usually inject 129 ES cells into C57Bl/6 blastocysts to generate chimeras.

You should provide us with an ampoule of low passage targeted cells that we can culture and inject.

Pronuclear injections
You should provide us with DNA at a concentration of 3 ng/μl.

DNA should be purified and linearized.

We will provide at least 3 positive mice.

For Pronuclear Injection of DNA:

We need ~500μl linearized DNA at a concentration of 3ng/ul.

We will provide you with purified tail DNA for analysis from all mice born. You should let us know within 2-3 weeks which mice to keep as founders.

If we are to analyze offspring, you should provide us with primers and PCR conditions.

Genotyping
You should provide us with PCR primers and PCR conditions.

Karyotyping
We will stain and count at least 50 spreads.
You should provide us with one ampoule of cells.

Breeding mice

Cryopreservation by freezing sperm
Cryopreservation of Mouse Spermatozoa

Cryopreservation by freezing embryos
You should provide us with at least 5 males and 10 females/mouse line.
Embryo Cryopreservation

IVF

Thaw mice from frozen eggs

Rederivation

Assay ES cell clones by PCR

Assay ES cell clones by Southern blot

Target construction