Transgenic Gene-Targeting Mouse Facility

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
Price: $5,000-$5,500

Gene Targeted Mouse Production
Production of mice using blastocyst injection of targeted ES cells
Price: $5,500-$6,000

Transgenic Mouse Production
Production of mice using pronuclear injection of DNA
Price: $3,500-$4,500

CRISPR Mouse Production

Production of mice using injection of CRISPR’s into early embryos.
Price: $3,500-$5,000

Validation of guideRNA
Injection or electroportion of CRISPR guideRNA’s to determine efficiency of cutting before proceeding to mouse generation
Price: $500-$750

Cryopreservation of Mouse Sperm
Preservation and storage of mouse sperm using liquid nitrogen
Price: $550

IVF
In vitro fertilization to rederive mouse lines from frozen or fresh sperm
Price: $1,000-$1,500

Rederivation of Mouse Lines
Rederivation of mouse lines from fertilized eggs or blastocysts
Price: $800

Cryopreservation of Mouse Embryos
Preservation and storage of mouse embryos using a controlled rate freezer
Price: $1,500

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
Price: $150

ES Cell Line Derivation
Derive embryonic stem cell lines from your mice
Price: $2,000

Karyotyping
Determination of chromosome number of ES cells
Price: $250

N.B.: These prices reflect our services for University of Utah researchers. Other non-profit organizations or universities will be charged 1.5 times the above prices. Private industry will be charged 2 times the above prices.

Contact: Susan Tamowski 801-581-3437 tamowski@genetics.utah.edu

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.

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

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.

TK1TK2A

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

FRT EM7 neoFRT

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

pACN

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, et.al., Genes and Development 13:1524, 1999.)

pACN Sequence

Jackson Labs
http://www.jax.org/

MGI Mouse Genome Informatics
http://www.informatics.jax.org/

International Society of Transgenic Technology
http://www.transtechsociety.org/

University of Michigan Transgenic Animal Core
http://www.med.umich.edu/tamc/index.html

Knockout Mouse Project
http://www.komp.org/

BACPAC Resource Center
http://bacpac.chori.org/

Email Contact: tgcore@cores.utah.edu
Phone Contact: 801-581-3437

Susan Tamowski, Director
801-581-3437
tamowski@genetics.utah.edu

Crystal Davey, Co-Director
801-585-0662
mutrus@genetics.utah.edu

Kyle O’Connor, Co-Director
801-585-7414
koconnor@genetics.utah.edu

He Lan
Senior Lab Specialist
801-581-3437
hlan@genetics.utah.edu

Nick Black
Lab Specialist
801-585-7414
nblack@genetics.utah.edu

Charles Murtaugh, Ph.D.
Co-Director of Transgenic & Gene Targeting Core
Chairman of Oversight Committee
Associate Professor
Department of Human Genetics
murtaugh@genetics.utah.edu

Kevin B. Jones, MD
Associate Professor
Huntsman Cancer institute
kevin.jones@hci.utah.edu

Suzanne Mansour, Ph.D.
Professor
Department of Human Genetics
suzi.mansour@genetics.utah.edu

Dean Tantin, Ph.D.
Associate Professor
Department of Pathology
dean.tantin@path.utah.edu

The objective of the Transgenic and Gene Targeting Mouse core is to provide access to mouse genetics technologies and thus enhance the research productivity of the University of Utah. Methods of manipulating the mouse embryo and its genome are technically demanding and costly and cannot be practically sustained in most laboratories of individual investigators. Thus, the Transgenic and Gene Targeting Mouse Core brings these technologies to researchers within the University who otherwise would not have on-campus access to these powerful genetic methods.

As Director of the University of Utah Transgenic and Gene Targeting Mouse Core, I am committed to providing state-of-the-art outstanding services to University of Utah researchers as well as to investigators throughout the world. Our Core consists of four excellent research technicians who are able to perform any aspect of creating transgenic or gene targeted mice, cryopreserving sperm and embryos, performing IVF (in vitro fertilization), ICSI (intracytoplasmic sperm injection), laser assisted IVF. Our lab has two microinjection stations and now a third teaching station, two cell culture stations, a controlled-rate embryo freezer, several sophisticated microscopes, as well as a microforge, pipette puller, and a beveller to make our own injection needles.

We provide personal consultation regarding your research project, can provide suggestions to optimize your goal towards generating a valuable mouse, and are usually able to start your project within a few weeks of you giving us DNA. We have a very fast turn around and are willing to work with you one-on-one to get optimal results.

Susan Tamowski has worked in the field of gene targeted mice since 1986 when she was an intricate part of assisting Dr. Mario Capecchi to generate the world’s first gene targeted mouse (for which he won the 2007 Nobel Prize in Physiology). She has been involved in all aspects of generating both gene targeted and transgenic mice from constructing the targeting and transgenic vectors, to genetic analysis of mice, to phenotyping analysis, as well as all aspects of embryonic stem cell culture, embryo injection and mouse surgery. Susan has been Director of the University of Utah Transgenic and Gene Targeting Mouse Core since 2005.