GEM Transgenic Facility

Cores/Services

A.  Vector Design Core

The Vector Design Core will meet with the investigator to discuss the gene(s) of interest and the goals of the research.  Based on the scientific goals and working in close collaboration with the investigator, the core will develop a comprehensive plan to generate the necessary construct.  The technical services provided by the core include generation of gene-targeted vectors (knockout/mutant/conditional) via traditional cloning techniques, long PCR from isogenic DNA, or BAC-based recombineering; and generation of promoter/tissue specific transgenic vectors.

B.  Embryonic Stem Cell Core

The ES Cell Core laboratory will transfect commercially available ES cell lines (129 and C57) with the investigator’s targeting construct and isolate/expand antibiotic resistant colonies in 96-well plates for freezing and genomic DNA preparation.  The core will also screen the antibiotic resistant colonies for gene-targeted colonies by Southern blot.  Alternatively, the PI can screen colonies in his/her laboratory using genomic DNA provided by the core.  Once positive clones have been identified, the core will expand and stock the correctly targeted ES cell clones and prepare clones for microinjection.  The ES cell core can also propagate and stock targeted ES cell clones obtained from other sources.

C. Microinjection/Mouse Production Core

The microinjection/mouse production core offers four different services:  production of transgenic mice via DNA pronuclear injection; production of gene-target mice via blastocyst injection of embryonic stem cells; embryo cryopreservation; and line rederivation.  

DNA pronuclear microinjection: Using the validated linearized DNA construct prepared by either the GEM Vector Design Core or the investigator, the Microinjection/Mouse Production Core will create founder transgenic mice on a C57BL/6 background.  ICR, FVB and C57BL/6 hybrid backgrounds can be produced if investigators are willing to pay for the acquisition of stud males.

Embryonic stem cell/blastocyst microinjection: The core will inject targeted ES cell clones propogated/supplied by the Stem Cell Core or investigator into C57BL/6 blastocyts.

Embryo cryopreservation: Female animals will be superovulated, mated with practiced stud males and the resulting fertilized eggs cryopreserved.  Cryopreserved embryos will be stored in liquid nitrogen in two physically separate locations.  When requested, the cryopreserved embryos will be transferred to pseudopregnant recipients to restart the line.  With the exception of pseudopregnant females, all animals are derived from the investigator’s breeding colony, unless other arrangements are made.

Line rederivation: Line rederivation can be performed for animals arriving at the facility with an unacceptable health status.  Female animals will be superovulated, mated with practiced stud males, and the resulting fertilized eggs transferred to pseudopregnant recipient females.  With the exception of pseudopregnant females, all animals are derived from the investigator’s breeding colony, unless other arrangements are made.

D.  Genotyping

The GEM Facility will isolate genomic DNA from investigator provided tissue/cell biopsies.  Genotyping can be done by standard PCR, long-range PCR, and/or Southern blot analyses genotyping.  The Vector Design Core can assist investigators with the development of genotyping assays.

E.  Consultation

The Co-Directors are available to consult with investigators regarding all aspects of the generation and utilization of genetically modified mouse models.

F.  Education

The core will provide three educational opportunities for faculty, staff, and students: a series of college wide seminars, a monthly workshop, and a graduate level course.

Graduate Course:  The core offers a 3-hour graduate level course (VTMI 664) entitled “Strategies for Manipulating the Mouse Genome” in the fall semester of even numbered years.  The overall objective of this course is to provide students with the basic concepts and skills needed to design/generate constructs for mouse genome modification and to evaluate scientific literature that uses genetically modified mouse models.  Primary literature from diverse fields including genetics, toxicology, neuroscience, infectious diseases, developmental biology, gene expression, and pharmacology are used to illustrate the different technologies.

Goals and Organization

The goals of the Texas A&M University Comparative Medicine Program (CMP) Genetically Engineered Mouse (GEM) Facility are to:

• Develop genetically altered mice from concept to founders at a cost below that required for individual or commercial preparation

• Serve as a resource for information and methodology in DNA construct development, ES cell culture and screening, rodent reproduction, line preservation and rederivation

• Educate faculty, staff and students in the derivation/utilization of mouse models in biomedical research

• Serve as a repository for mouse lines

The facility is comprised of three cores: the Vector Design Core, the Embryonic Stem Cell Core, and the Microinjection/Mouse Production Core. These cores offer technical services (DNA and ES cell microinjection, DNA construct development, ES cell transfection/cloning, embryo cryopreservation and line rederivation); educational seminars and formal courses; and consultation with individual investigators. Investigators within the Texas A&M System and its affiliates have access to all of these services.

The day-to-day operation of the facility is the responsibility of the Co-Directors, Dr. Melanie Ihrig (Texas A&M University Comparative Medicine Program), Dr. Danna Zimmer (Texas A&M University Department of Veterinary Pathobiology) and Dr. Warren Zimmer (A&M Health Science Center Department of Systems Biology and Translational Medicine). The GEM Facility Advisory Committee develops policies and fee schedules for the facility. This committee is composed of representative members from Texas A&M University and the Health Science Center, as well as an outside member considered to be an expert in the field of genetically modified mouse models. Current members of the advisory committee include: Dr. Mary Meagher (Professor of Psychology, Texas A&M University); Dr. Emily Wilson (Associate Professor of Systems Biology and Translational Medicine, A&M Health Science Center); Dr. Jane Welsh (Professor Veterinary Biosciences, Texas A&M University); Dr. Phil Mirkes (Director, Center for Environmental and Rural Health, Texas A&M University); and Dr. Francesco DeMayo (Professor of Cell and Molecular Biology, Baylor College of Medicine).

Accessing Services

The first step in initiating a project is to meet with the core directors and submit a completed GEM Project Submission Form. The purpose of this meeting is to identify the core services that will be needed to meet the goals of the investigator’s project. A timeline and estimated costs for completing the project will be provided to the investigator.

A.  Vector Design Core

The Vector Design Core Laboratory can design and construct DNAs for any mouse genetic experiment. This includes knockout or conditional targeting vectors as well as generation of promoter specific transgene vectors. The specifics for each vector will be developed in consultation with the investigator. Each vector DNA is considered a separate project.

Investigator Responsibilities:

Step 1: Complete a GEM Project Submission Form and a Vector Design Core Project Form.
Step 2: Meet with the Core Director(s) to outline project and design the vector. The investigator will need to provide information concerning the project goals and basic genetic/expression data.  The strategy for completing the construction of the vector will be recorded on a GEM Consultation Worksheet that will be amended to the Vector Design Core Project Form. 
Step 3: Provide the genetic material (cDNAs, BACs, etc.) needed to start the project. Specific items will be dependent upon the services requested. 
Step 4: Provide additional information/guidance if technical problems arise.

Core Responsibilities:

In consultation with the Investigator, the Core will develop a strategy for constructing the vector. The investigator will receive weekly updates concerning progress and projected completion dates.  The core will create and keep a bound notebook for each project that contains the original data, protocols, notes and the weekly updates.  This notebook and the completed vector will be given to the investigator or another GEM core as deemed by the investigator.  The investigator will be given a glycerol stock of the completed vector and any intermediate cloning vectors.  The core will retain a second glycerol stock.

B. ES Cell Core

The ES Cell Core will transfect W4 embryonic stem cells with the investigator’s linearized construct; isolate/expand drug-resistant clones; identify/stock gene-targeted clones; and expand targeted ES clones for microinjection.  Investigators will be able to choose other ES cell lines (for example C57BL6) once the core has optimized procedures for working with these lines.

Investigator’s Responsibilities:

Step 1:  Complete a GEM Project Submission Form and an ES Cell Core Project Form.

Step 2:  Submit a Screening Assay Documentation Form.  The information provided on the Screening Assay Form ensures that correctly targeted clones will not be discarded due to technical problems with the Southern blot screening strategy. Because the life-span of ES cells frozen in a 96 well format is 30-60 days, it is imperative that the screening procedure be developed prior to targeting.  This information also ensures that subsequent breeding of the most appropriate founder lines will occur expeditiously and at minimal cost to the investigator. We also strongly recommend appropriate in vitro expression assays prior to submission. The core will provide the ES cell genomic DNA in a 96-well format and positive-control hybridization probes needed to develop genotyping assays. The GEM Vector Design Core will supply this information for any constructs they generate and can assist investigators with development/optimization of screening assays.

Step 3: Provide the core with 500 mg of construct DNA and a completed Plasmid Submission Form.  The investigator will be notified within one-week if the DNA meets transfection standards and if so an electroporation date will be set. The GEM Vector Design Core will provide plasmid DNA for all constructs that it develops.

Core Responsibilities:

The core will provide control hybridization probes and 96-well format genomic DNA required for documentation of screening assays. The core will linearize, purify, and electroporate the investigator’s construct into W4 embryonic stem cells. Resistant colonies will be cloned and expanded into triplicate 96 well plates —2 plates for genomic DNA preparation and 1 plate for recovery of gene-targeted clones. Southern blot/PCR analyses will be used to identify gene-targeted clones. The core will expand, re-verify, and stock 4 vials for each investigator-selected clone. Two vials will be delivered to the investigator and two vials will be kept in the core for microinjection. The core does not guarantee a minimal targeting efficiency. In addition, there is no guarantee that targeted clones will generate chimeras and/or germ-line transmission.

C. DNA Pronuclear Injection

Using the validated linearized DNA construct prepared by either the GEM Vector Design Core or the investigator, the Microinjection/Mouse Production Core will create founder and/or transient transgenic mice on a C57BL/6 background. ICR, FVB and C57BL/6 hybrid backgrounds can be produced at an additional cost.

Investigator Responsibilities:

Step 1: Complete a GEM Project Submission Form and a Microinjection Project Form.

Step 2: Submit a completed Screening Assay Documentation Form and reagents needed for identifying founders including oligonucleotide probes and control DNA. These materials insure that transgenic animals are not accidentally discarded because of technical problems with the tail DNA purification and/or PCR assays. Although not required, investigators are strongly encouraged to submit documentation of the Southern blot analysis that will be used to assess copy number, number of integration sites and transgene integrity. The GEM Vector Design Core will supply this information for any constructs they generate and can assist investigators with development/optimization of screening assays.     

Step 3: Provide the core with enough plasmid DNA to liberate 10µg of insert DNA upon restriction digestion and a completed Plasmid DNA Submission Form. The GEM Vector Design Core will provide plasmid DNA for all constructs that it develops. The investigator will be notified within one week if the DNA meets microinjection standards and if so, a microinjection date will be scheduled for 2-3 weeks later.  This will allow ample time for ordering, acclimating and superovulating the female donor mice.

Step 4: Vivarium personnel will transfer transgenic founders to the investigator’s AUP/animal room following weaning and serological testing of the recipient dam (approximately 56 days post injection).  The investigator is responsible for breeding and testing the founder lines.  For transient transgenics, the investigator will receive pregnant mothers 13 days after injection, unless other arrangements have been made.  A Cage Hand-Off Form will accompany all transient transgenic recipients.  The information on this form is essential for monitoring the productivity of the GEM Facility and should be returned to core personnel when genotyping results are available. Additional projects will not be initiated for investigators with outstanding forms.

Core Responsibilities: 

The Core will microinject >150 zygotes with the DNAconstruct and reimplant surviving embryos into pseudopregnant recipient females. The injections will be spread over two nonconsecutive days and recorded on Microinjection/Stem Cell Worksheets. Tail biopsies will be processed 30 days after injection. Positive founders (pups with the transgene incorporated into the genome) are not guaranteed. However, in the absence of positive founders, additional injection days can be scheduled at a reduced cost.

D. Embryonic stem cell injection into blastocysts

The Microinjection/Mouse Production Core will inject gene-targeted embryonic stem cell clones prepared by the GEM ES Cell Core, or investigator, into C57BL/6 mouse blastocysts.

Investigator Responsibilities:

Step 1:  Complete a GEM Project Submission Form and a Microinjection Project Form.

Step 2: Submit six or more targeted ES cell clones to the GEM ES Cell Core Facility. Because chimerism and germ-line transmission are clone-dependent, investigators are strongly encouraged to submit multiple clones for injection. Injection dates will be approximately one-month after receipt of targeted clones. This allows for acquisition, acclimation, and superovulation of donor mice as well as expansion of the ES cell clones.

Step 3: Vivarium personnel will transfer chimeric F0 founders to the investigator’s animal room following weaning and serological testing of the recipient dam. This will be approximately 65 days after the microinjection date. The investigator is responsible for breeding the chimeras and subsequent confirmation of germ-line transmission.

Core Responsibilities: 

The Core will inject each clone into 20-30 3.5 day C57BL/6 blastocysts and reimplant embryos into pseudopregnant recipient females. All injections will be recorded on the Microinjection/Stem Cell Worksheets. The core guarantees only that a minimum number of blastocysts will be injected with each ES cell clone. If chimeras are not generated, the core will work with the investigator to obtain germ-line competent targeted ES-cell lines and chimeras in a cost-efficient manner.

E. Embryo Cryopreservation:

Female animals will be superovulated, mated with practiced stud males, and the resulting fertilized eggs cryopreserved and stored in liquid nitrogen in two physically separate locations.

Investigator’s Responsibilities:     

Step 1:  Complete a GEM Project Submission Form and a Cryopreservation Project Form.

Step 2:  The investigator will provide a minimum of four practiced male studs and four 3-4 week old females are required. Males will be returned to the Investigator’s colony and females will be euthanatized. To maximize the yield of fertilized embryos, stud males must be singly housed for a minimum of 1 week, but no more than 2 weeks, prior to the scheduled cryopreservation date. The investigator will receive Cage Card Tags to be placed on cages during the initial consultation.

Step 3:  The Investigator will receive a copy of the Cryopreservation Worksheet indicating the number of embryos/straws frozen and their location. 

Core Responsibilities:

The core staff will superovulate females, mate them with practiced studs, isolate embryos, identify fertilized embryos, and freeze 40-60 embryos/straw. Due to the variability in fertility between mouse lines/strains, the core does not guarantee embryo yield. In instances of low embryo yield, the core will work with investigators to optimize fertility and achieve cryopreservation with minimal expense to the Investigator. 

F. Line Rederivation

Female animals will be superovulated, mated with stud males, and the resulting fertilized eggs transferred to pseudopregnant recipient females.

Investigator’s Responsibilities: 

Step 1:  Complete a GEM Project Submission Form and a Line Rederivation Project Form.

Step 2:  In consulation with the core, determine the number of transfers to be performed.

Step 3:  Provide the core with the required number of stud males and donor females.  Males will be returned to the Investigator’s colony and females will be euthanatized. To maximize the yield of fertilized embryos, stud males must be singly housed for a minimum of 1 week, but no more than 2 weeks, prior to the scheduled mating. The investigator will receive Cage Card Tags to be placed on cages during the initial consultation.

Step 4:  Vivarium personnel will transfer weaned pups to the Investigator approximately 3 weeks after rederivation.   

Core Responsibilities:

The core staff will superovulate females, mate them with practiced studs, isolate fertilized embryos, and transfer fertile embryos to pseudopregnant females.

G. Genotyping

The core will prep genomic DNA from tissue/tail biopsies and perform PCR or Southern blot analyses.  Both approaches include positive control single copy genes. Genotyping is performed on a first-come first-served basis. Turn around time for PCR analyses averages 5 working days for Southern blot analyses 2-3 weeks.

Investigator’s Responsibilities:

Step 1:  Complete a GEM Project Submission Form and a Genotyping Project Form.

Step 2:  Five working days prior to biopsy, inform the core of the delivery date, number, and type of samples. This ensures that biopsies will be processed immediately upon receipt.

Core Responsibilities:          

The core staff will purify genomic DNA and perform the genotyping requested.

H. Consultation

The Co-Directors are available to consult with investigators regarding all aspects of the generation and utilization of transgenic and knock-out animal models. Interested investigators should contact one of the Co-Directors.  All consultations are recorded on a Consultation Worksheet.