Transgenic Core Facility Services
Transgenic Core Facility Services
University Committee on the
Care and Use of Animals
Prior to the production of genetically altered animals, investigators
must have approval to use mice in their research from the University Committee on
Use and Care of Animals.
Fees
This facility exists to serve the needs of University of Michigan
researchers. A list of fees is availalbe.
Investigators are invited to contact Thom Saunders with any questions.
The genetically altered animals provided by
this facility can only be used for research purposes. Access to cell
lines and plasmids is contingent upon approved material
transfer agreements. Request forms are
available for download.
Sample
Preparation
For the production of conventional transgenic mice, investigators are
responsible for providing a restriction digest containing 50 ug of the
transgene insert. The Transgenic Core will purify DNA
for microinjection
as described. A minigel photo should accompany the digest, the DNA
fragment to be purified should be clearly marked. Several things should
be considered in designing a transgenic research project (see Transgenic Project
Outline). Prokaryotic vector sequences interfere with the
expression of some transgenes, thus unique restriction sites at the 5'
and 3' ends of the construct should be available for vector removal.
The transgene should contain unique markers so that its presence can be
easily detected in DNA samples and its expression can be assayed and
distinguished from endogenous gene expression.
Certain transgenes may result in a very low yields of transgenic founders due to the intrinsic nature of the transgene. For example, certain genes will be deleterious or incompatible with proper growth and development of the embryo. Special arrangements should be made with the Core if the transgene is suspected to cause lethality. The expression of a transgene requires that the appropriate transcriptional control elements be included in the DNA construct. Expression is often influenced by the chromosomal site in which the transgene DNA is integrated. Preliminary studies in cell cultures are recommended to verify the integrity of the construct and the function of the promoter/enhancer. However, it is not always possible to predict in advance whether the transgene will be expressed in vivo. For these reasons, the Core cannot guarantee that transgenic founders will express the transgene.
For the production of gene targeted mice with mutations induced by homologous recombination in ES cells, investigators are responsible for producing ES cell clones with targeted genetic mutations. The first step in gene targeting is to obtain a detailed restriction map of the genetic locus and knowledge of exon/intron boundaries. (see Gene Targeting Outline) Higher yields of targeted ES cell clones are obtained from isoegenic DNA, thus a clone(s) from a genomic library that exactly matches the ES cell line to be electroporated is strongly advised. Restriction mapping data is used to identify 5' and 3' arms for targeting vector construction and to establish a screening strategy for the identification of ES clones that have undergone homologous recombination. This requires the characterization of probes for Southern blot analysis that lie outside of targeting vector sequences. After the targeting vector has been cloned it is introduced into ES cells. Fastidious cell culture technique and specialized reagents are required to maintain ES cells in a pluripotent state. Differentiated ES cells will not produce chimeras with the ability to transmit targeted mutations through germ cells. Investigators are invited to contact Elizabeth Hughes for training in ES cell culture and to obtain reagents certified for ES cell culture. Due to the intrinsic variability of individual ES cell clones we can not guarantee that germline chimeras will be produced from any particular ES cell clone. The Transgenic Facilty tests and validates ES cell culture reagents to maximize the successful outcome of gene targeting projects.
Services Offered: View Detailed Service List
2. Bacterial Artificial Chromosome (BAC) Recombineering Services: The Transgenic Facility includes a BAC Recombineering Core. BACs containing genes of interest are obtained from BAC library resources. Homologous recombination of DNA fragments is used to modify BAC sequences so as to produce gene knockins, gene knockouts, piont mutations, or to generate gene targeting vectors. Specialized bacterial strains and plasmid reagent have been obtained from the NCI for this purpose. Additional plasmids useful for recombineering are under development in the BAC Core. Modified or native BACs can be used as transgenes in the generation of BAC transgenic mice or BAC transgenic rats. BACs of any species for which the complete sequence is known can be modifed. Contact Michael Zeidler or Thom Saunders with any questions.
3. Gene Targeting Service: This is a collaborative service combining the knowledge of the investigator with the specialized skills of the Transgenic Core. This service produces embryonic stem cells with mutations induced by homologous recombination with a targeting vector provided by the investigators. The stem cells can be used to generate germline ES cell-mouse chimeras and mice with novel mutations. Some may wish to conduct in vitro differentiation studies. It allows investigators to focus on the molecular biology pf the gene in question while the Core focuses on generating pluripotent embryonic stem cells with the targeted mutation. This collaborative approach emphasizes the strengths of each partner and obviates the need for laboratory personnel to master the fastidious technique that is necessary to culture totipotent mouse embryonic stem cells. Contact Thom Saunders with any questions.
The investigator performs the following tasks: Restriction map the endogenous gene. Establish a robust, reliable, reproducible screen that discriminates between wild type and targeted alleles. Provide experimental data demonstrating that the Southern blot probes confirm the predicted genomic structure of targeted ES cell clones. Clone the gene targeting vector or make arrangements with the BAC Recombineering Core to prepare the targeting vector - contact Contact Thom Saunders for BAC Core information. Purify gene targeting vector DNA for electroporation according to the protocol supplied by the Transgenic Core. Analyze genomic DNA from as many as 480 ES cell clones for homologous recombination with the targeting vector. Timely analysis is important because of the limited life span of ES cells cryopreserved at -80 degrees C. Review the screening data with the Transgenic Core prior to expansion of cryopreserved clones.
Transgenic Core staff will provide/perform the following information/procedures: Prepare an experimental time line for planning purposes. Electroporate the targeting vector into ES cells. Pick 480 electroporated ES cell clones (five 96-well plates). Each plate of the five 96-well plates will be split into three. Two plates will be cryopreserved in independent -80 degree C freezers. One plate will be grown and split into two replicates for DNA preparation. Prepare DNA from replicate 96-well plates (ten plates total, two replicates of each of the five plates of clones). Deliver the DNA plates to the investigator for screening.
Once ES cell clones with targeted mutations are identified, Core
staff will continuously expand ES cell clones until 5 vials of 5 X 106
cells/ml can be produced for storage in liquid nitrogen. Each clone
will
be tested for mycoplasma and morphology evaluated. Clones will be
chromosome counted to identify euploid clones and ES cell pellets will
be provided to the investigator
DNA extraction and
verification of targeting. Clones that meet the criteria for
microinjection
(euploid, good morphology, clear of infection, correctly targeted) can
be
scheduled for microinjection into blastocysts for chimera production.
4. ES
Cell-Mouse Chimera Production: The Core will inject C57BL/6
blastocysts with embryonic stem cells (129 mouse derived ES cell
clones) or albino C57BL/6 ES blastocysts (C57BL/6 ES cell clones). A
minimum
of 60 blastocysts will be injected with each ES cell clone. Orders must
be canceled two weeks in advance in order to avoid a cancellation fee.
The
Core will purchase the required mice and pay for their housing,
including
vasectomized males and females for foster mothers. The mice are housed
under
specific pathogen free conditions. Blastocysts will
be collected, injected with 10 to 16 ES cells, and transferred to
pseudopregnant
recipients. When the chimeric pups are three weeks old the Core will
transfer
the mice to the investigator. If desired the investigator can make
arrangements with the Transenic Core for germline breeding of ES
cell-mouse chimeras.. The
generation of chimeric mice is highly dependent on the condition of the
ES cells. Thus, we can not guarantee germline chimeras will be produced
from each ES cell clone. Investigators interested in culturing
their own ES cells for blastocyst microinjedion are encouraged to
contact Elizabeth
Hughes regarding ES cell lines and their culture requirements.
5. DeNovo
ES Cell Line Derivation. The Transgenic Core will prepare
new
mouse ES cell lines from blastoycsts or mice provided by investigators.
Standard methods employing serum containing medium and MEK1 inhibitor
are used. The success rate of this procedure is very high (100% success
to date) as long as blastocysts can be obtained from the mouse strain
in question. Mouse ES cells provide a endless supply of cells for in
vitro studies because ES cells do not undergo senesence and cease
division as do other cell types (e.g. fibroblasts). In addition is
possible to differentiate ES cells into different lineages to query
gene function in cell culture systems instead of or in parallel to in
vivo studies. The procedures requires intensive cell culture over an
extended period of time by Transgenic Core personnel.Advance notice
should be given, preferably when the blastocyst-donors-to-be are born.
The ideal age for of in-house blastocyst donors for superovulation
response is 24-28 days. A request form can be downloaded here. Contact Thom
Saunders well in advance to schedule this procedure. Training
in ES cell culture is available for those who
are interested. See Personnel Training.
6. Personnel
Training: Training in the production of genetically altered
mice
and ES cell culture are offered:
Pronuclear Microinjection Training: Individuals will
be trained in two stages: first, embryo collection and reimplantation,
and
second, microinjection of fertilized eggs with transgene DNA. BAC
Transegnic inject training is available upon request. 40 hours of
training is provided. Transgenic Rat training is available in addtion
to transgenic mouse production training. 40 hours (1 week) of training
is provided. It is not unusual for investigators to generate
transgenic founders during the training week.See the sample Mouse Syllabus and/or the
sample Rat Syllabus.
Blastocyst Microinjection Training: Individuals will
be trained in two stages: first, embryo collection and reimplantation,
and
second, microinjection of blastoycsts with ES cells. 40 hours (1 week)
of training is provided. See the sample Syllabus.
Mouse ES Cell Training Class: Those
with
prior experience in handling mice will require less time. Individuals
will be trained in all aspects of ES
cell
culture and manipulation. They will learn every technique necessary to
successfully
produce ES cell clones with targeted mutations induced by homologous
recombination. Investigators
are expected to present relevant papers and discuss papers that provide
the experimental basis for procedures learned in the laboratory setion
of the class. 80 hours (two weeks) of training is provided. See
the sample Syllabus.
Contact Thom
Saunders to
schedule training.
7. Plasmids, ES Cells, and Culture Reagents: Plasmids and ES cell reagents are maintained by the Core. The following plasmids are available: pnlacf, which contains beta galactosidase as a reporter gene; pPNT, a plasmid for the construction of gene targeting vectors; the pflox vector designed for the preparation of mouse models with tissue specific gene inactivation. A CMV-Cre plasmid (pBS185) is commercially available from Addgene. The Core has numerous germline competent mouse ES cell lines available from both 129 mouse strains and C57BL/6 mouse strains. Others can be obtained through KOMP. In addition, the Core tests fetal bovine serum (FBS) for lots which will support ES cell proliferation with minimal differentiation and stores a large quantity for resale at cost. Feeder cells for ES cell culture are prepared from neor transgenic mice to optimize culture conditions during positive selection. Please contact Thom Saunders with any questions on how to initiate material transfer agreements for these reagents.
8. Centralized Support: This consists of access to microinjection and ES cell culture workstations. Microinjection support includes quality tested media for egg and blastocyst culture, quality tested hormone stocks, equipment maintenance, and all of the miscellaneous plastic and glass supplies. Users will be responsible for maintaining their own animals for embryo donors, male studs, vasectomized males, and pseudopregnant females. Plasticware for tissue culture of ES cells is provided at cost. Users will be responsible for providing their own media.Pluripotent ES cells, tested FBS, and feeder cells are available from the Core at nominal cost (see 4. above). Please contact Thom Saunders to schedule equipment use.
9. Conversion of Mouse Lines to Specific Pathogen Free Status: The Core will obtain fertilized eggs from pathogen infected mice and transfer them into clean, specific pathogen free (SPF) recipients as described (Van Keuren and Saunders, 2004). The investigator is responsible for providing mice of the strain in question. The Core will transfer washed, fertilized eggs to SPF pseudopregnant females. The mice will be tested to verify the SPF status of the offspring. This method eliminates viral (e.g. murine hepatitis virus, Sendai virus, parvovirus), bacterial (e.g. mycoplasma pulmonis), ectoparasite (mites), and endoparasist (pinworm) infections. We have converted many different mouse strains to SPF status by this procedure. Our success rate is 100%, in every case the foster mothers and offspring have been free of infection. Please contact Thom Saunders if you need to convert mice to pathogen free status.
Van Keuren ML, Saunders TL. 2004.Rederivation of Transgenic and
Gene-Targeted Mice by Embryo Transfer. Transgenic Res. 13:363-371.
10. Embryo Cryopreservation/Recovery: The purpose of embryo freezing is to protect against the loss of valuable, unique mouse stocks through breeding failure or disease, and to eliminate the cost of maintaining mouse lines not actively in use. Investigators will provide the Transgenic Core with stud males and egg donors from the line or stock which they desire to preserve. Typically 15 stud males are mated weekly with egg donors per cryopreservation session to produce embryos for cryopreservation. On average, it takes 4 embryo collection sessions to freeze down enough embryos to guarantee recovery. If you can provide us with homozygous males then fewer sessions will be needed. However, it may take more sessions if the mouse strain has a low superovulation rate, the stud males have low fertility, or males are too old. Some strains can not be successfully cryopreserved. The investigator is responsible for providing all stud males, embryo donors, and per diem costs for these animals. The Core will collect and freeze fertilized mouse eggs at the eight cell stage. A test batch of embryos will be thawed and transferred to pseudopregnant recipients from each cryopreservation session. Frozen embryos will be maintained in liquid nitrogen and an annual storage fee will be assessed. Please contact Galina Gavrilina (or 763-6209) if you wish to cryopreserve or recover stocks.
How much does it cost?
Our recharge is based on a set fee per
cryopreservation session. A cryopreservation session means that we
collect embryos from
15 donors, freeze down 8-cell embryos, test thaw some of the embryos,
culture the thawed 8-cell embryos to blastocyst, transfer the
blastocysts to recipients, and count then number of fetuses or pups
that result. We may ask you test DNA from the pups to verify their
genetic composition.
How many cryopreservation sessions does it take to freeze a line?
Assuming that you have 15 singly housed hemizygous transgenic mice and
you are using C57BL/6 egg donors it will take about 3 or 4 sessions to
store 600 cryopreserved embryos (300 transgenic embryos) in liquid
nitrogen. If the stud males are homozygous for the gene then fewer
sessions will be needed since we can stop after we freeze down 300
embryos. If the males have poor fertility and we obtain fewer than 100
embryos per cryopreservation session, it will be more cost-effective to
replace the males than to do 6 or more sessions to generate the needed
embryos to cryopreserve your line.
Why do you need to freeze so many embryos?
This standard is widely adopted by commercial providers. Our goal is
to freeze down the embryos and guarantee that we can bring the mice
back
from cryopreservation in the future. This lets you euthanize all of the
mice on the shelf in your mouse room while being assured of access to
the
animals for your future research.
What if we can't generate 300 transgenic/knockout embryos to
cryopreserve my mouse strain?
Our goal is to bank 300 embryos with the desired genotype. This
allows for long-term storage of your embryos in multiple sites for the
highest level of security and recovery. If we are unable to generate
300 embryos because of poor reproductive performance we may still be
able to recover your mice from cryopreserved embryos once or twice,
depending on the number of embryos frozen and their performance on
thawing.
Who covers the animal purchase and per diem costs?
You do. Galina Gavrilina
(763-6209)
will sit down with the person who is managing your mouse colony and
work
out a schedule for mouse deliveries. You need to provide 15 fertile
stud
males and order in a series of egg donors for superovulation and mating
to the studs. Galina will collect the 8-cell embryos after mating,
freeze
them down, and test thaw them.
Can I go ahead and do a freeze if I have only 6 to 12 males?
Based on our experience, this is costly, inefficient, and very time
consuming. With so few males available, fewer eggs for cryopreservation
will be produced each session. This means that a greater proportion of
the eggs will be lost to test thaws. Simply put, we need you to provide
15 stud males for us to go forward.
What if I have an FVB/N transgenic line that I want to freeze down?
We can freeze down FVB/N lines. However, it may require more than 3 or
4 sessions because FVB/N female mice produce fewer eggs than C57BL/6
eggs in response to the superovulation treatment.
11. Speed
Cryo
Speed Cryo is a method to cryopreserve mouse strains. Cryopreservation
safeguards mouse strains against breeding failures, pathogens, and
genetic contamination. Speed Cryo relies on in vitro fertilization
(IVF) to generate 2-cell embryos for cryopreservation. Our goal is to
bank 300 embryos with the
desired genotype. Once a strain is banked, un-needed live stocks
of mouse can be eliminated.
Compared to the standard
approach of cryopreserving 8-cell embryos, Speed Cryo requires
fewer males to generate embryos for cryopreservation. A single session
with 2-4 homozygous males and C57BL/6 egg donors can generate 300
embryos for cryopreservation. If heterozygous males are used, it is
likely that two or more Speed Cryo sessions will be needed to produce
300 embryos for cryopreservation. The
advantage of Speed Cryo is that many embryos can be generated from a
single
IVF procedure. The disadvantage of the Speed Cryo approach is that it
depends
on good IVF yields. The efficiency of mouse IVF varies depending on the
strain
background and the fertility of the individual males used as sperm
donors
(Byers, et al., 2006, Vergara et al., 1997). Thus, it is possible that
IVF with transgenic
or
knockout sperm will produce few eggs for cryopreservation even though a
control
IVF with hybrid mouse sperm gives good results. In these cases, it is
advisable
to repeat the Speed Cryo procedure to control for variability in the
transgenic or knockout males. If too few eggs are generated
then the remaining option is generate and freeze down 8-cell
embryos.
Vergara
GJ, Irwin MH, Moffatt RJ, Pinkert CA. 1997. In vitro
fertilization in mice: Strain differences in response to superovulation
protocols and effect of cumulus cell removal. Theriogenology
47:1245-1252.
The Transgenic Core will superovulate 25 C57BL/6 egg donors (other
strains can be substituted) perform IVF with sperm from the males you
donate to us and 20 C57BL/6 egg donors. We will do a positive IVF
control with sperm from (C57BL/6 X DBA/2)F1 male and 5 C57BL/6 egg
donors. After overnight culture, 2-cell embryos will be
frozen stored in liquid nitrogen. When sufficient numbers of embryos
are present, they will be divided between two liquid nitrogen
containers in two different buildings. We will perform a test
thaw on each batch of cryopreserved embryos. Thawed embryos will be
scored
for survival and transferred to pseudopregnant females. The
females
will be scored for pregnancy and the tissue from the pups will be
provided
to the investigator. We expect the investigator to genotype the pups
and
determine whether the pups have the desired genotype.
Contact Galina Gavrilina
(or 763-6209) to schedule
Speed Cryo procedures. Download Speed Cryo Request
Form.
Speed Cryo Cost
We expect investigators to provide sperm donors and to pay for
the purchase of egg donors. In
addition to the service fee you will be
recharged for the purchase costs of the egg donors. The complete fee
covers the IVF
procedure, cryopreservation of 2-cells eggs, storage in monitored
liquid nitrogen vessels, and a test thaw and transfer of frozen eggs to
verify viability.
12. In
Vitro Fertilization
Mouse In Vitro Fertilization (IVF) can be used to rapidly expand mouse
lines from a few males that carry the desired genotype or
to
maintain strains with poor breeding efficiency. The Transgenic
Core
will superovulate 25 C57BL/6 egg donors per session (other
strains can be
substituted).
We will perform IVF with sperm from your males and 20 C57BL/6 egg
donors.
We will perform a positive IVF control with sperm from (C57BL/6 X
DBA/2)F1
males and 5 C57BL/6 egg donors. After overnight culture, 2-cell
embryos
will be transferred to pseudopregnant females. All weaned pups
will
be transferred to the investigator. We expect the investigator to
genotype
the pups and determine which pups have the desired genotype(s).
IVF results vary according to genetic
background and the quality of
individual males used for IVF (Byers et al., 2006, Vergara et al.,
1997). Thus we can not
offer a guarantee that any given IVF procedure will produce large
number of pups. The standard recharge for each IVF session is the cost
of
purchasing the egg donors and a fee to recover
our costs.
If both the experimental and the control
IVF procedures fail then we will not recharge your account.
Contact Galina Gavrilina
(or 763-6209) to schedule IVF procedures. Download IVF Request Form.
13. Sperm
Cryopreservation
Mouse sperm cryopreservation is offered as a routine service. Request forms are available. We will
collect sperm from your male(s) and freeze down 10 aliquots per male
and store them in liquid nitrogen. We recommend that you order an IVF
service to test the sperm aliquots for their ability to generate viable
pups with the desired genotype. Recovery of live mice from frozen mouse
sperm is strain dependent (Thornton et al., 1999).
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