ES Cell Electroporation |

ES Cell Electroporation

Introduction

Gene-targeted mouse ES cells are produced by electroporating a gene targeting construct into wild-type ES cells. The targeting construct typically contains selectable markers surrounded by two regions of homology to the targeted locus. In a fraction of those cells that take up the targeting construct, homologous recombination between the genome of the ES cell and the two regions of homology will result in the replacement of the targeted locus with the targeting construct.

Knock-out or knock-in mice are created by microinjecting these gene-targeted mouse ES cells into mouse blastocysts. The injected blastocysts are then transferred to pseudopregnant females and allowed to develop to term. The resulting pups are chimeras.

Service Description

The gene targeting service includes the following:

electroporation of your targeting vector into wild-type mouse ES cells
culture of cells under antibiotic selection
picking up-to 96 drug-resistant clones
splitting clones into two sets of 24-well plates-one set will be frozen at -80°C and the other will be provided to the investigator for genotyping
thaw and expand targeted ES cell clones
provide investigator with a 10 cm confluent analysis plate for Southern Blot Analysis (one plate for each clone carrying a homologous targeting event)
freeze and store duplicate clones in LN2 up to 5 years for future experiments/microinjections

Targeting Construct Design

Gene targeting constructs are designed to undergo homologous recombination into a specific locus chosen by the investigator, usually with the aim of disrupting the gene to prevent transcription of a functional mRNA (a knock-out), or mutating the gene (a knock-in). There are many ways to design a targeting construct. Below are some general considerations:

In order to ensure that no protein is made from the targeted gene, construct a targeting vector that will delete as much of the coding sequence as possible.
The arms of homology should be cloned from the same strain as the targeted ES cells. For most projects, we recommend targeting to C57BL/6.
The longer the arms of homology, the better. A common strategy to aid in genotyping is to design one arm at 5-6 kb and the other arm at 1-2 kb for an overall length of 7 kb.

Gene Targeting Vector Preparation

It is the investigator’s responsibility to design and engineer the targeting vector. The TRP offers free consultations for targeting vector and genotyping design.
The investigator should provide 30-50µg of linearized targeting construct DNA at a concentration of 1 µg/µl.
The DNA should be absolutely free of contamination from ethidium bromide, ethanol, phenol, chloroform, and high salt.
Design a genotyping protocol (Southern Blot Analysis and/or PCR) that can distinguish homologous recombination events from random integration events. This step should be completed before submitting your DNA vector for electroporation. As an aid for establishing a reliable genotyping protocol, the TRP highly recommends that all investigators prepare a control vector which includes the area outside of the region of homology.

ES Cell Lines Available

The TRP uses the G4 ES cell line for gene targeting experiments.

G4 ES cells: This line was created by Andras Nagy from a hybrid of 129S6/SvEvTac x C57BL/6Ncr. This is our preferred line for gene targeting experiments due to its ability to consistently produce high-percentage germline-capable chimeras. This line is available to all University of Washington investigators with a signed MTA.

Prices and Guarantees

The charge includes ES cell transfection, drug selection, picking clones, plates for storage at -80° C, duplicate plates for analysis, thaw and expansion of potentially targeted clones for freeze at -196° C and one 10cm plate per targeted clone for Southern Blot Analysis, and storage of confirmed targeted ES cell clones in LN2 for 5 years. Because different selection vectors included within the targeting construct result in widely variable enrichment for targeted events, no guarantees as to the number of clones arising from electroporation can be made.

It is the investigator’s responsibility to establish a reliable genotyping protocol (PCR and/or Southern Blot) before the electroporation process is started. Clones must be screened promptly. ES cell clones that are stored at -80° C for more than 1 month will not be guaranteed to produce high-percentage chimeras.
Click here for current rates.

Schedule an ES Cell Electroporation Session

To schedule an ES cell electroporation session, go to our TRP Forms page. Fill out a “Request for ES Cell Electroporation” form and deliver it along with your gene targeting construct to Serina Tsang.