The Transgenic and Gene Targeting Mouse Core is available to make transgenic mice using pronuclear injection of DNA constructs including BAC DNA into embryos. The Core is able to generate gene-targeted mice using CRISPR technology to directly inject reagents into early embryos resulting in quick production of targeted mice. We also use traditional knockout or conditional constructs electroporated into embryonic stem (ES) cells followed by blastocyst injection of targeted ES cells to obtain germline chimeric mice eventually leading to the production of gene-targeted mice. The Transgenic and Gene Targeting Mouse Core offers related procedures including cryopreservation and storage of mouse sperm and mouse embryos, rederivation of mouse lines from frozen embryos, in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to rederive frozen sperm, development of primary ES cell lines from targeted mouse lines, and karyotyping of ES clones.

The Core is available for technical advice regarding injection procedures, cell culture techniques, vector design and construction, as well as analysis of ES cell clones and mice. The Core is amenable to new ideas and available to try new methods that researchers are interested in. The Core maintains the necessary mouse colonies for basic procedures, including a large colony of C57Bl6 mice, albino C57Bl6 mice, C57Bl6 x CBA F1 mice. We also maintain a small colony of ROSA-flp mice in the C57Bl6 background, and CMV-cre deleter mice. The Core maintains 129 ES cells, 129/C57Bl6 hybrid ES cells, and C57Bl6 ES cells (N and J) for gene-targeting. Equipment includes three full microinjection stations and dissection microscopes with fluorescence imaging capabilities, Femtojet injectors, Peizo drills an XYClone laser, pipette pullers, microforges, and IVF incubator, and a rate controlled embryo freezer.

The Core has successfully provided genetically altered mice to many University of Utah researchers as well as to researchers throughout the world. The Core has rederived mouse lines for researchers using frozen sperm and frozen embryos shipped from around the world.

Please acknowledge the University of Utah Transgenic and Gene Targeting Core in publications and grant proposals resulting from work that was done in our Core. Published acknowledgments are tracked by NIH and can impact grant funding for the entire University.


The cores are frequently called upon to validate their contributions to the University community and its research endeavors. One way we do that is through the number and quality of publications utilizing the cores. Your acknowledgements help us gain the recognition we need to continue to be supported by the University. Thank you for helping us so we can continue to serve you.


Example acknowledgement text:

We would like to acknowledge the University of Utah Transgenic and Gene Targeting Core for providing transgenic and/or gene targeted mice that were used in this research.



Gene Targeted ES Cell Production
Production of gene targeted mouse embryonic stem cells


Gene Targeted Mouse Production
Production of mice using blastocyst injection of targeted ES cells


Transgenic Mouse Production
Production of mice using pronuclear injection of DNA


CRISPR Mouse Production

Production of mice using injection of CRISPR’s into early embryos.


Validation of guideRNA
Injection or electroportion of CRISPR guideRNA’s to determine efficiency of cutting before proceeding to mouse generation


Cryopreservation of Mouse Sperm
Preservation and storage of mouse sperm using liquid nitrogen


In vitro fertilization to rederive mouse lines from frozen or fresh sperm


Rederivation of Mouse Lines
Rederivation of mouse lines from fertilized eggs or blastocysts


Cryopreservation of Mouse Embryos
Preservation and storage of mouse embryos using a controlled rate freezer


Exportation of Embryos or Sperm
Collect live embryos to transfer to mice to another facility. Send embryos or sperm in a liquid nitrogen dry shipper


ES Cell Line Derivation
Derive embryonic stem cell lines from your mice


Determination of chromosome number of ES cells

DNA Purification for Injection (Transgenic)

Purification of DNA for Microinjection

1. Perform a restriction digest to release the gene of interest from vector sequences.

2. Separate the insert of interest from the vector on an agarose gel run in TAE (not TBE).

3. Excise the gel slice containing the gene fragment of interest and elute the DNA.

4. Purify the DNA either by using a DNA purification column or by phenol-chloroform extraction and ethanol precipitation.

5. Final DNA should be dissolved in low EDTA-TE injection buffer (10mM Tris/0.1mM EDTA pH 7.5). This buffer must be prepared with Milli-Q water to maintain zygote viability. We will need 1-2ug of DNA at a concentration of at least 100 ng/ul.

6. Determine the DNA concentration. Leave the DNA in concentrated form in TE injection buffer.

7. The core facility will dilute your sample for injection.

Preparation for Targeting Vector for Electroporation (Gene-Targeting)

The goal is to have 50-100 µg of purified, linear plasmid DNA at a final concentration of approximately 1ug/ul.

1. Grow and purify plasmid DNA.

2. Digest plasmid DNA with appropriate enzymes to linearize and to cut out vector DNA

3. Extract DNA with pheno/chloroform, precipitate with ethanol and re-suspend in TE.


4. Purify using DNA purification column.

5. Resuspend final purified, linearized DNA in TE (allow overnight at 4°, followed by vigorous ‘flicking’).

6. Measure concentration and 260/280 ratio.

ES Cell Preparation for Injection (Blastocyst Injection)

Give us a frozen vial of cells that we can thaw, grow and prepare for microinjection.


Contact us regarding a date and time for injections and you can bring us live freshly prepared cells for injection. Cells must be at a concentration of 4×105 cells/ml in microinjection medium.

From ES Cell to Mouse: A Brief Description

From ES cell line clone to homozygous knockout mouse

After electroporation of your construct and selection of ES clones we will give you duplicate 96-well plates containing 5-10ug of DNA from each of about 200 clones. You will analyze these clones using PCR and/or southern blot to determine which have homologously recombined in the correct location on the chromosome (targeted). Usually 1-20% of the clones will be targets.

We can karyotype your best lines, and then inject two of these lines of ES cells into blastocysts. We usually inject 8-12 ES cells (129-derived or 129/Bl6-derived which are agouti in color) into each C57Bl6 (black in color) blastocyst. We implant about 100 injected blastocysts into several pseudopregnant foster mothers over the course of one week of injections. Of the pups born, we generally get at least 5 good chimeric males that are at least 80% agouti. This indicates that most of the mouse is made up of the injected targeted ES cells rather than the black host.

We then breed these chimeric males back to C57Bl6 females to check for germline transmission. Agouti offspring indicate that the injected ES cells made germ cells as well as somatic cells, demonstrating germline transmission. Of these agouti pups half will carry the targeted mutation (since coat color is on a different allele than your gene). If you have made a straight knockout, you will get a homozygous knockout by mating two of these targeted heterozygous offspring together. If you have made a loxed mutation, you will have to mate these loxed heterozygotes to a cre driver mouse.This first generation will only make a heterozygote for your loxed out mutation (since the cre mouse has a wild type allele), so you have to mate two cre positive, loxed out allele positive mice together to get your mutation homozygously loxed out in the cre specific tissue.

Timing: It takes about a year from DNA construct to loxed out homozygous mouse with a phenotype.

Time from DNA to Knockout Mouse (Table)
Vectors Available

These vectors are obtainable from the University of Utah Transgenic and Gene Targeting Mouse Core and may be used with the permission of Dr. Mario Capecchi.


Clone your entire targeting vector into this TK1TK2A vector to improve efficiency of targeting by using negative gancyclovir selection in addition to neo positive selection. There is a multiple cloning site between the two TK’s.

TK1TK2A Sequence


For generating a conditional knockout, the pFENF contains a neo cassette flanked by frt sites for easy removal by exposing to flp recombinase. Multiple cloning sites are outside the frt sites for ease of insertion of arms and addition of lox sites.

FRT EM7 neoFRT Sequence


For use in making a straight knockout, the pACN vector contains a self excising neo cassette with cloning sites outside each lox site for homologous arms. The testes specific promoter driving cre deletes neo in the male germ cells. (Bunting,, Genes and Development 13:1524, 1999.)

pACN Sequence

Useful Links

Jackson Labs

MGI Mouse Genome Informatics

International Society of Transgenic Technology

University of Michigan Transgenic Animal Core

Knockout Mouse Project

BACPAC Resource Center

Email Contact:
Phone Contact: 801-581-3437

Transgenic & Gene Targeting Mouse Core

Crystal Davey, Co-Director

Kyle O’Connor, Co-Director

He Lan
Senior Lab Specialist

Nick Black
Lab Specialist

Oversight Committee

Charles Murtaugh, Ph.D.
Chairman of Oversight Committee
Associate Professor
Department of Human Genetics

Kevin B. Jones, MD
Associate Professor
Huntsman Cancer institute

Suzanne Mansour, Ph.D.
Department of Human Genetics

Dean Tantin, Ph.D.
Associate Professor
Department of Pathology