OHSU

Services & Costs

Transgenic Mouse Models Fees  (01/01/2013-06/30/2013)


Transgene Microinjections

Code     
Service                                                 
Standard Price
101B B6D2F1 hybrid                                                
$3,700
101B1
B6D2F1 hybrid (One Day Injection) $1,650
101C C57BL/6 $4,800
101C1 
C57BL/6 (One Day Injection)                               
$2,100


ES Cell Gene Targeting

Code Service Standard Price 
102 ES Cell Gene Targeting $4,200
102A
Expanding ES cell clones from 96-well plates     
 
102A1
1 clone
$550
102A2
2 clones
$700
102A3
3 clones
$850
102A10  
Additional Clone, up to 10 clones
$150 each additional
102B
Expand one vial of ES cells to freeze back
$500

 Blastocyst injection/Chimeras

Code      
Service                                                                  
Standard Price 
103A
1 Clone into B6 blasotcysts
$2,450
103A1
One day injection into B6 blasotcysts
$1,550
103AB
1 Clone into B6-albino blasotcysts
$2,550
103AB1 
One Day Injection into B6-albino blasotcysts                          
$1,600

Rederivation of Mouse Line

Code Service Standard Price
104B
Rederivation via IVF with Frozen-Thawed Sperm
$1,500
104A     
Rederivation via Embryo Transfer
$550

Cryopreservation of Germplasm

Code Service Standard Price
110E
Cryopreservation of Embryos
$1,650
110EQC
Quality Control of Frozen Embryos

by Retransfer in Uterus

$200
110S
Cryopreservation of Sperm
$500
111 Cryo Storage of Embryos / Sperm

1 line / year

$100
111A
Cryo Storage of ES cells

1 line / year

$100
112 Generation De Novo mutant ES Cells from

Genetically Modified Mice

$2,100


Supplements

Code Service Standard Price
120A        
ES Cell Media, 500 ml, Complete (handling fee)
$95
120B
Wild Type ES cells (1 vial)
$200
120C
MEF feeder cells (1 vial, 5 x 10^6 cells)
$250

Standard Prices may vary depended on mouse donor strain and client's affiliation. Changes will be based on the cost of additional reagents, mice and time of personnel of TgMMSR.

 

Miscellaneous Service

 Miscellaneous Service means different types of additional services e.g. isolation of mouse tissues, breeding etc. Prices of Miscellaneous Service will be based on the cost of additional reagents, cells, mice and time of personnel of TgMMSR.

Code
Service
Standard Price
121D
Mouse Cage Per Diem - Large Cage
$0.96 / day
121A
Hourly Rate
$42.00 / hr
121B
Material Variable
121C
Mouse Cage Per Diem - Small Cage
$0.75 / day

 

pronuclear microinjection

 

Background

The first transgenic animals were produced more than 30 years ago (Jaenisch & Mintz, 1974; Gordon et al., 1980) and now transgenic technology is successfully and broadly used in different fields of biomedical research. Animals can be defined as transgenic when they have integrated foreign DNA into their germ line as a consequence of experimental introduction of DNA (Palmiter & Brinster,1985). Although several approaches exist to introduce foreign DNA into mammalian genome the majority of transgenic mice are produced by microinjecton of plasmid DNA into pronucleui of fertilized eggs. Traditionally to achieve stable integration and expression a transgene is injected as a several kilobases  fragment of plasmid DNA. As result it can integrates in random regions of different chromosomes as concatemer (several copies of transgene) and thus the founders may have different levels of transgene expression. 

Many different modifications of transgenic technology were developed at the past. For example, to achieve single copy integration of transgene and expression at physiological level in the appropriate cell types a transgene together with regulatory elements can be delivered to the mouse genome as a Bacterial Artificial Chromosome (BAC) or Yeast Artificial Chromosome (YAC) containing several hundred kilobases from mouse or human genome (Giraldo, Montoliu, 2001; Van Keuren et al., 2009). Since majority of injected genes are expressed the technique is generating exciting opportunities to study differential gene function, abnormal gene regulation in malignant disease and mutations of genes cause developmental effect. Moreover, the use of for example tet-inducible (Freundlieb et al., 1997; Fedorov et al., 2000) and/or Cre/Lox conditional (Schönig et al., 2002; Wang, 2009) transgenic systems provides an ability to control the mammalian gene expression in a temporal and spatial manner particularly during development and oncogenesis (For more infoormation, see useful Links and References).

 New Flaten Transgene compressed

Generation of transgenic mice  (A) Isolation of the 7 kb transgene (yellow arrow) on an agarose gel after digestion of a plasmid by HindIII and XhoI enzymes. (B) MicroinInjection of a transgene into the pronucleus of a fertilized mouse egg. (C) PCR identification of transgenic founders (+) among newly generated progeny. (D) Tet-inducible expression of a beta-gal reporter gene in the roof of the hindbrain and spinal ganglia (yellow arrows) of tTACMV/NZL double transgenic embryos at E12.5. (E) Lateral overview of a "Cherry" transgenic embryo at E15.5. (F) "Cherry" expression in the skeleton of the embryo. The transgenic mice were produced for Dr. R. Schweitzer (Shriners Hospital, OHSU, Portland, OR).

Resources and Recommendations

Transgenes

Standard plasmid transgenes: The transgene DNA produced by the customer can be sent as either intact supercoiled plasmid DNA or as a purified injection-ready transgene insert. To obtain high quality DNA the purification of the plasmid for microinjection can be done by centrifugation in a CsCl gradient. Other purification options also exist as many laboratories are successful using DNA purification kits as simple and fast tools to achieve microinjection quality DNA.

 

Preparation of DNA for Pronuclear Injection

1. Purification of supercoiled plasmids by one of these methods:

a) Centrifugation in a CsCl gradient.

b) UltraClean™ GelSpin™ Kit (MoBio Laboratories, Cat. No 12400-250).

c) GenElute Plasmid Miniprep Kit (Sigma Cat. No NA0150).

2. Cut the injection fragment out of the cloning vector using the appropriate restriction enzymes. Final yield should be 10 - 20 micrograms of transgene fragment.

3. Separate restriction digest products on an agarose gel using either TBE or TAE buffer. Use either low- or standard- melting temperature agarose. Make sure the gel is run long and slow enough to isolate the fragment from the vector.

4. Place gel on a transilluminator.  Use a clean razor blade or scalpel to cut out the transgene fragment.  Minimize the time of exposure to UV-Light.

5. Use the Qiaquick gel extraction kit from Qiagen (Cat. No. 28704). Do not use Vortex if your fragment is larger than 10 kb.

6. Quantitate DNA solution.

7. Verify size the intact condition of the DNA on a minigel.

8. Adjust the concentration of the purified transgene to 50 - 100 ng/µl and submit frozen DNA sample containing at least 1 µg of DNA to TgMM.

9. Transgenic Core will dilute, aliquot and store the eluted DNA at -200 C.

 

If TgMM is asked to purify the transgene insert, please provide 50 - 100 µg from a maxi-prep from Qiagen or CsCl-purified plasmid DNA with instructions for isolating the transgene fragment. These instructions must be accompanied by a copy of the gel showing the size of the fragment.

BAC transgenes: BAC transgenes can be injected into one of our standard strains of mice as indicated below; either in the F1-hybrid strain (B6D2F1), or the inbred C57BL/6 strain. For BAC transgenic mice, please purify the DNA by CsCl centrifugation, and send as linearized DNA at a concentration of approximately 50 – 100 ng/µl. Prior to injecting the BAC transgene we will dilute and equilibrate the DNA in a buffer containing salt and polyamines.

Micro Inject Setup

Microinjection setup mounted on anti-vibration table 

Mouse Strains

Inbred C57BL/6 transgenic mice: Currently the majority of our customers request transgenic mice on the C57BL/6 inbred background because the genome and phenotype of this mouse line is well known. This background is also advantageous if the transgenic line is crossed with induced mutants that are maintained as congenic strains on this background. We offer the production of transgenic mice on the C57BL/6 inbred background with either plasmid or BAC transgenes. However, because the overall efficiency is lower in C57BL/6, we charge higher fees than the F1-hybrid transgenic mice.

F1-hybrid mouse strain: The C57BL/6 x DBA/2 F1 hybrid strain has been used in this core for standard transgene microinjections. Under the terms of the agreement, we will produce at least 30 offspring from the injections of pronuclear stage embryos. These animals will be transferred to the PI's colony at 3 weeks of age (weaning age).

Responsible Parties and Timeline

Step

Timeframe

Protocol

Responsible Party

1

variable

project inception, vector design

customer

2

variable

preparation/submission of transgene construct

customer/TgMM

3

variable (depends on # of projects)

facility schedules injection

TgMM

4

week 1

microinjection of transgene DNA

TgMM

5

week 4

offspring born

TgMM

6

week 7

transfer potential offspring to customer

customer

7

variable

genotype pups

customer

8

variable

breed transgenics and genotype F1s

customer



Service Ordering

Before initiating a transgenic mouse project, please receive approval of the Animal Care and Use Committee (IACUC) and the Institute Biosafety Committee (IBC) for your animal use and recombinant DNA protocols. Then fill out, sign and submit the Project Request Form to TgMM.

In addition to the Project Request Form, please provide the following materials:

1) A map of the construct and a simplified description of the phenotype you expect to produce.

2) A restriction digest containing approximately 50 micrograms of your plasmid.

3) A photo of an aliquot of the digest analyzed by agarose gel electrophoresis with the transgene band clearly identified.

 

Service fees include:

1) Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).

2) Superovulation of the virgin females.

3) Microinjection into 250-300 C57BL/6 or B6xDBA-2 zygotes.

4) Transplantation of the microinjected embryos into pseudopregnant recipient females.

5) The production of 3 guaranteed transgenic founders. TgMM does not guarantee expression of the transgene  

 

Service fees do not include:

1)  Animal housing charges for litters after weaning.

2)  Ear/tail tissue collection for DNA isolation for genotyping.

3)  Health testing of the foster mother.

4)  Shipping charges.

5) Animal housing charges of recipients and their offspring until weaning. Customers will be charged per diem according to DCM pricing ($.075 per cage/day) for the timespan the foster mothers are with their litters while remaining within the Transgenic Core's mouse room.

 

 Additional Services:

According to the customers' request, TgMM can purchase and use other mouse lines for the generation of genetically modified mice. Moreover, TgMM can tag, collect tail samples, extract DNA and genotype the offspring by PCR.  For this purpose the animals can be housed in our colony beyond the usual 3 weeks (weaning age). These additional services will be charged as extra fees, including the extra costs for the cage(s) per diem.  

 

Service fees:

See Fees for Transgene Microinjections.

Gene Targeting in ES Cells

 

Background

Intense development of molecular genetics and embryology at the end of the last century made it possible to develop basic new methods of mutagenesis called "Gene Targeting" (Thomas & Capecchi, 1987; Capecchi, 2005). Mario Capecchi, Martin Evans and Olive Smithies were declared laureates of the 2007 Nobel Prize in Physiology or Medicine for Gene Targeting. These technologies use genetically-encoded vectors (linearized plasmids) as directed mutagens in embryonic stem (ES) cell lines. Mouse ES cell lines are  pluripotent  stem cells and are first obtained through cultivation of the inner cell mass of mouse blastocysts of the inbred line 129/Sv (Evans & Kaufman, 1981; Martin, 1981). These cells can remain in an undifferentiated state for a long time under certain conditions of in vitro cultivation. ES cells can colonize all tissues after their reintroduction into preimplanted embryos. 

Knock out and Gene Trap vectors per se are additional techniques for gene inactivation. The inclusion of two homologous regions of preliminarily chosen genes and Neo and Tk genes (for positive and  negative selection respectively) in a knockout  vector allows the knockout vector to target and  integrate into the investigators region of interest.  Another variant of vector mutagenesis also based on the employ of ES cells, is gene trapping. Gene trap vectors have no homologous fragments with genomic DNA therefore they can integrated into random genomic loci and cause mutations and simultaneously incorporate a reporter gene. The main elements of most gene trap vectors are two genes: the lacZ  reporter gene  encoding beta-galactosidase that allows histochemical visualization, and the gene for neomycin phosphotransferase (Neo)  providing for cell resistance against geneticin (G418) selection. Introduction of Knock-out or Gene Trap vectors into genome of ES cells by electroporation or retroviral infection results in inactivation of certain genes in vitro. Currently, researches exploit both methods to inactivate genes of interest depending on the specifics of their studies. Moreover, modifications of these methods combined with Cre/LoxP and/or FLP/Frt - based recombination systems provide the ability to inactivate genes of interest in tissue specific and/or time specific manner (Kühn et al., 1995; Wang, 2009). Furthermore, ES cells are employed in other fields of transgenic technology, for example to produce transgenic cells and mice with one copy of transgene  (via Rosa 26 approach) with predictable level of expression. To facilitate these procedures the International Knock out Gene Trap (www.komp.org) and EUCOMM (www.eucomm.org) consortiums generated thousands different modifications of mutant ES clones available for the international scientific community (See more in Useful Links and References).    

ES genetarg[1] 

Gene targeting in ES cells. (A) Colonies of JM8.A ES cells derived from C57BL/6N mice cultured on a feeder layer of    mouse embryonic fibroblasts (phase contrast). (B) BTX electro square porator. (C) Expression of beta-galactosidase reporter gene in Gene Trap ES cell clone produced via an electroporation of a Gene Trap vector.

Resources and Recommendations

 ES Cells

Several ES cell lines on the 129/Sv and 129xC57Bl/6 background are available for targeted mutagenesis in TgMM. To prevent differentiation of ES cells, they are cultured on the special media containing LIF. The cells have a high capacity to colonize germline of chimeric mice and were successfully used to generate many novel knock out mouse lines.

Mouse Embryonic Fibroblasts (MEF)

To support ES cells growth we have wild type MEF on CD-1 and C57Bl/6 background. To perform ES clones selection after transfection we have multiple-drug resistant MEFs prepared from the Tg(DR-4) mouse strain (Tucker et al., 1997)   from Jackson Lab that displayed resistance to concentrations of the drugs G418, 6-thioguanine, puromycin and hygromycin well above those used normally for the selection of drug-resistant ES cells.    

Targeting

To transfect ES cells please provide 50 µg of DNA comprising the targeting construct, linearized and precipitated in EtOH. The   targeting    construct DNA will be electroporated with a batch of 2x107 cells from the requested ES cell line. Following drug-selection, ~ 300 ES cell colonies will be isolated and replica plates generated, which will be forwarded to the PI's laboratory for genotyping. The master plates will be stored at - 170° C freezer for further analysis and the production of chimeric mice.

Karyotype Analysis

Prior the blastocyst injections, we strongly recommend karyotype analysis for ES cell clones to check for euploidy, which is essential for germline transmission. If a clone is partially aneuploid, subcloning may be warranted before blastocyst injections are initiated. ES cells are analyzed by the OHSU Cytogenetics Core Laboratory using G-banded metaphase spreads. Generally, the Cytogenetics Core provides a report from the analysis of 20 metaphase spreads. Please fill out a separate Cytogenetics Request Form for each clone to be analyzed and forward to the Transgenic Core. The clones will be grown in Petri plates in the Transgenic Core and submitted to Cytogenetics Core for analysis.

Responsible Parties and Timeline

Step

Timeframe

Protocol

Responsible Party

1

variable

project inception, project design

customer

2

variable

preparation of targeting construct, validation of targeting assay

customer

3

week 1

preparation of feeder cells and ES cells including medium for growth and selection

TgMM

4

week 2

electroporation of targeting construct DNA

TgMM

5

week 2

selection, picking and growing ~300 colonies in 96 well plates

TgMM

6

week 3

splitting cells into multiple 96-well plates for Southern/PCRanalysis, shipping frozen cells to customer

TgMM

7

variable

analysis of ES cell clones for targeted replacement of desired locus

customer



Service ordering

Fill out, sign and submit the Project Request Form to TgMM.

In addition to the Project Request Form, please provide the following materials:

1)  A map of the construct and a simplified description the phenotype you expect to produce.

2)  Fifty µg of the targeting construct, linearized and precipitated in EtOH.

3)  A photo of an aliquot of the digest analyzed by agarose gel electrophoresis, with the transgene band clearly identified.

 

Service fees include:

1)  Preparation of ES cells and mouse embryonic fibroblast feeder (MEF) cells.

2)  Electroporation of targeting vector into ES cells.

3)  Selection and picking of ES clones on four 96 well plates.

4)  Preparation and freezing of eight replica plates.

5)  Supply of replica plates for molecular analysis by investigator.

6) Cryo Storage of master and replica plates through 6 months.

 

TgMM does not guarantee homology recombined clones.

 

Service fees do not include:

1)  Design of the gene targeting construct and preparation of DNA for electroporation.

2)  Isolation of DNA from positive clones and molecular analysis of the targeted clones.

3)  Shipping frozen cells to external customer.

 

Additional services:

According to the customer's request TgMM can expand positive clones from 96-well plates, freezing positive clones in vials and supply of clones aliquots for additional genotype confirmation. Moreover, TgMM can provide wild type ES cells, wild type and drug resistant MEF feeder cells. These additional services will be charged extra fees.

 

Service fees:   

See Fees for ES Cell Gene Targeting. 

Generation of ES Cell Derived Chimeras

Background

Introduction of mutant ES cells into developing embryos is an alternative way to generate novel genetically modified mouse lines. The transfer of mutant ES cells into pre-implanted embryos and subsequent transplantation of chimeric embryos in the uterus of a pseudopregnant female does not affect their development which leads to the birth of a normal viable progeny. During embryogenesis, ES cells can colonize all tissues of the chimeric embryo, including the germ cell lineage (germline) when the differentiated ES cells become involved in spermatogenesis of the adult chimeras. The resulting chimeras will transmit mutation to their offspring and as a result novel mutant mouse lines will be produced. Hence, ES cells serve as a bridge between in vitro genetic manipulations and the in vivo consequences of these manipulations. One of main requirements for a successful application of the gene targeting technology is the capacity of ES cells to give origin for gametes (germline formation) in a chimeric organism. It is known that long term culture of ES cells leads to the block of germline transmission (Nagy et al., 1993; Fedorov et al., 1997) that apparently results from accumulation of spontaneous genetic or epigenetic changes of the ES cell genome. Therefore it is necessarily to keep ES cells under optimal cell culture conditions and to perform a minimal number of passages.
Various methods were established to introduce ES cells into host embryos, these include: injection into the blastocyst cavity (Moustafa, Brinster, 1972), injection under the zona pellucida of morulas (Lallemand, Brûlet, 1990) and aggregation with a morula (Wood et al., 1993). Injecting into the blastocyst cavity is now the most commonly used approach for the production of ES cell chimeras.

New Chimeras

Generation and analysis of chimeras. (A) Injection of ES cells into a blastocyst. (B, C, D) Chimeric mice derived from mutant ES clones with the genetic background of 129/Sv, C57Bl/6 agouti and 129/Ola respectively. Chimeras (B, C and D were produced by injection ES cells (129/Sv background) into C57BL/6 B6 blastocysts for Dr. J. Christian (University of Utah) and Dr. A Barnes (OHSU, Portland, Or.). Respectively all chimeras demonstrated high intensity of agouti coloration of the fur and further germline transmission. Chimeras (B,C,D,E) were produced by the OHSU TgMM within 2009-2012. (E, F) Chimeric mice were produced by injection of ES clones (C57Bl/6 agouti) into C57BL/6-Albino blastocysts for Dr. E. Tucker (West Virginia University School of Medicine) and Dr. P.G. Barr-Gillespie (OHSU, Portland, Or). Chimeras (E, F) were produced by the OHSU TgMM within 2012. (G) Expression of green fluorescent and red fluorescent reporter genes in distinct subpopulations of cells in the pituitary of a POMC/GFP-POMC/RFP aggregation chimeric mouse produced for Dr. M.J. Low (University of Michigan). (H) The limb specific expression of the beta-gal reporter gene in gene trap embryos at E9, E9.5, E10.5 (upper row, left to right), and E11.5 (lower row), respectively. (I) Ubiquitous expression of the beta-gal in gene trap embryos at E9.5 (Project of TgMM, OHSU).

Resources and Recommendations

Currently we perform blastocyst injections of ES clones that were either produced by ourselves or our customers. Furthermore, we are actively using gene trapped and gene targeted ES cell clones produced by the International Knockout and Gene Trap Consortiums (see Useful Link and References). The Consortiums have genetically engineered ES cell clones on the 129 and C57BL/6 backgrounds. At this time our specific attention is focused on the ES clones with a C57BL/6N genetic background. To produce new mutant mouse line we recommend the purchase of 2-3 different clones, with the price being approximately $500-$700 per one ES cells clone. The generation of a new mouse line will cost $4000-$6000 including the cost of ES clone, delivery of ES cells and the generation of chimeric mice via blastocyst injection.

It is known that ES cell lines accumulate chromosomal abnormalities during culture that reduce or completely abolish the chance of generating of germline chimeras (Robertson, 1987; Longo et al., 1997).Therefore we, in collaboration with the Cytogenetics Core, are able to improve the quality of your mutant ES clones by a selection of euploid subclones. We are also capable of eliminating differentiated ES cells and improving the germline transmission capacity of your mutant ES clones by culturing and subcloning under special conditions (for example, culture with RESGRO or ESGRO-2i Medium, Millipore).

Responsible Parties and Timeline 

Step

Timeframe

Protocol

Responsible Party

1

variable

analysis of ES cell clones for targeted replacement of desired locus

customer

2

week 1

grow up 2 independent clones of ES cells confirmed to be targeted.  Microinject each clone into 8-- 100 blastocysts in order to produce chimeric offspring.

TgMM

3

week 4

offspring born

TgMM

4

week 8

transfer chimeras to customer

customer

5

week 10

mate chimeras to identify germline transmitting mice

customer

6

week 15

genotype germline transmitting animals

customer

Service ordering

1)  Before initiating a transgenic mouse project, please receive approval of the Animal Care and Use Committee (IACUC) and the Institute Biosafety Committee (IBC) for your animal use and recombinant DNA protocols.

2)  Fill out, sign and submit the Project Request Form to TgMM.

3)  If the ES cells were not produced by TgMM please provide the following materials:  

a) ES cells and information about the origin of these ES cells.

b) Information about the health status of ES cells.

c) Protocol for handling of the ES cells.

 

Service fees include:

1)  Purchase of virgin C57BL/6 or B6xDBA-2F1 females (donors of embryos).

2)  Expansion of positive ES cell clone for injection into blastocysts.

3)  Injection of cells of one ES cell clone into 80-100 C57Bl/6 or C57BI/6-albino blatocysts.

4)  Transfer of microinjected embryos into pseudopregnant recipients.

5)  Generation of 15-20 pups.

6)  If we use ES clones generated by us, we guarantee the generation of 4-5 chimeras with 50% agouti on the fur. However, we don't guarantee germline transmission.

 

Service fees do not include:

1)  Animal housing charges of litters after weaning.

2)  Breeding of chimeras for germline transmission. 

3)  Health testing of the produced mice.

4)  Shipping charges.

5) Animal housing charges of recipients and their offspring until weaning. Customers will be charged for the time when forster mothers with litters will remain in Transgenic Core's mouse room per diem, according DCM price ($0.75 per cage/day).

 

Additional services:

According to the customer's request, TgMM can perform a germline transmission assay. For this purpose the animals will be housed in our colony beyond the 3 weeks of weaning age. Furthermore, Transgenic Core can generate aggregation chimeras. Moreover, TgMM can tag, collect tail tissue samples, extract DNA, and genotype the offspring by PCR. These additional services will be charged as extra fees including the additional costs for the cage(s) per diem.

 

Service fees:   

See Fees for Blastocyst injection/Chimeras. 

Embryo and Sperm Cryopreservation

 

Background

Exponential growth of mutant mouse lines and the number of animals required for definitive studies has  led to exponential growth in animal costs and challenges for colony management. Cryopreservation of mouse germplasm (embryos and sperm) is an efficient and cost effective solution for managing colony size. In addition, this service permits archiving of transgenic models not currently needed but potential invaluable to future studies and provides protection against spontaneous mutations occurring in subsequent generations maintained in the live colony. Cryopreservation of mouse strains has traditionally relied on freezing embryos at the 2-cell or 8-cell stage, mainly because live mice can easily be reconstituted by embryo transfer (Whittingham et al., 1972).  

An alternative to embryonic cryopreservation is sperm cryopreservation followed by in vitro fertilization (IVF) (Critser, Mobraaten, 2000).  However, reconstituting live mice by IVF from frozen-thawed sperm is inefficient with some strains, notably with the C57BL/6 strain, the most common background on which induced mutants are bred (Sztein et al. 2000). The main problem of C57BL/6 sperm is that frozen-thawed spermatozoa are inactive, due to a requirement of an incubation within the female tract for the activation.  Moreover, the spermatozoa of frozen sperm samples have no or reduced motility often, therefore reconstituting of a mouse strain can only be achieved by an intracytoplasmic sperm injection (ICSI) into unfertilized oocytes (Wakayama, Yanagimachi, 1998; Li et al., 2009).

The most effective method of cryopreservation of a mouse line is the Speed Cryo approach (Byers et al., 2006). The method consists of superovulation of virgin females, following isolation of unfertilized oocytes and subsequent IVF using freshly collected sperm from males of the mutant strain. Following  fertilization and culture,  2 or 8 cell  embryos can be frozen using the standard method. Ideally only 3-4 males are required to prepare and freeze 250-350 embryos. However, the efficiency of Speed Cryo depends on the effectiveness of IVF, strain background and the fertility of the individual sperm donors (Vergara et al., 1997; Byers, et al., 2006).

4CryoTools 1 

Cryo-preservation accessories used in our laboratory. (A) Eight cell embryos prepared for cryopreservation. (B) Plastic accessories listing from top to bottom: concave cryogenic vials used for freezing ES cells, a conical cryogenic vial used for freezing of embryos/sperm/oocytes, cryogenic straws used for freezing of embryos/sperm/oocytes. (C) FTS controlled Freezer for embryo cryopreservation. (D, E) Taylor-Wharton extended time liquid nitrogen refrigerator and cryostorage system used for long term storage of germplasm. 

4IVF 

Development of mouse embryo after in vitro fertilization. From left to right:  IVF process, 2-cell stage embryo, 8- cell morula, and blastocyst.

Resources  and Recommendations

Embryo Cryopreservation:  For strain cryopreservation, 10-12 homozygous or heterozygous stud males will be mated with superovulated wildtype females. Approximately 250-300 eight cell stage embryos will be collected and frozen.  Roughly 20-30 embryos will be stored per straw, which can then be stored in liquid nitrogen in the Core's  freezers. Freezing of 8-cell embryos is the most stable stage for embryo cryopreservation and storage. 

 

Sperm Cryopreservation: This is the most fast and least expensive option for cryopreservation of mouse strains. Frozen sperm can be stored in LN2 indefinitely, and reconstitution of live mice can generally be achieved using IVF. Following collection of epididymal sperm, 20 samples will be frozen and stored in liquid nitrogen. However, keep in mind that IVF is inefficient with frozen-thawed sperm from some strains such as C57BL/6, which has critical implications since many induced mutant strains are maintained as congenic strains on that background. Therefore for greater reliability during recovery, we recommend using our Speed Cryo service (see below) for freezing a strain on the C57BL/6 inbred background.

 

IVF Embryo Cryopreservation (Speed Cryo): We began implementing the Speed Cryo approach in 2011. The freshly collected sperm from 3-4 transgenic males is used to fertilize the eggs of superovulated females of the same strain background via in vitro and then cryopreservation of 2 cell or 6-8 cell stage embryos is done. Simultaneously we also freeze 10 samples of sperm from these males. Thus, your strain will be cryopreserved and stored as both embryos and as well as sperm.

Despite several approaches of cryopreservation existing we recommend to employ Speed Cryo for C57BL/6 and as well as for other mouse lines.

 

Intracytoplasmic Sperm Injection (ICSI): Currently we are in the process of establishing ICSI of frozen sperm into unfertilized oocytes.   

 

Quality Control: Each strain frozen by the standard method or Speed Cryo can be checked to ensure overall quality and viability of embryos using one of two methods:

1. Following standard or Speed Cryo freezing, one straw will be thawed, and the embryos will be placed in culture for development to blastocysts where a >75% development to blastocysts will be considered a successful cryopreservation. 

2. Following standard or Speed Cryo freezing, one straw will be thawed, and the embryos will be placed in culture for development to blastocysts followed by a uterine transfer into a pseudopregnant recipient. The generation of live pups displaying the appropriate genotype (genotyping to be done by the customer) will be considered a successful cryopreservation.

3. After sperm cryopreservation one straw will be thawed and a quality of the spermatozoa will be visually evaluated under a microscope.

 

Long-term Storage of Germplasm: The frozen embryos and sperms are stored in liquid nitrogen in two freezers located at two geographically distinct sites: one located at the OHSU Main Campus and the other at the West Campus. Each freezer is monitored by OHSU Public Safety using a remote alarm.

Responsible Parties and Timeline

Step

Timeframe

Protocol

Responsible Party

1

week 1

Providing of mutant stud males

customer

2

week 2

Embryo cryopreservation

TgMM

3

week 3

Thaw embryos and those quality control via transfer to foster mom

TgMM

4

week 6

Transfer newborn offspring to customer

TgMM

5

variable

PCR genotyping of newborn offspring

customer



Service Ordering

To initiate a freezing project, please fill out and forward a Cryopreservation Request Form to the TgMM.

In addition to the Project Request Form, please provide the following materials:

1)  For standard cryopreservation of 8-cell stage embryos provide 10-12 stud males at the age of 2-9 months.

2)  For Speed Cryo provide 3-4 stud males at the age of 2-9 months.

3)  For Sperm Cryopreservation provide 3-4 stud males at the age of 2-9 months.

 

Standard Embryo cryopreservation service fees include:

1)  Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).

2)  Cryopreservation of 250-300 eight-cell embryos.

3)  Cryostorage of frozen embryos for 1 month.

 

Speed Cryo cryopreservation service fees include:

1)  Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).

2)  Cryopreservation of 250-300 eight cell embryos.

3)  Cryopreservation of 10 samples of sperm.

4)  Cryostorage of frozen embryos and sperm for 1 month.

 

Sperm cryopreservation service fees include:

1)  Cryopreservation of 20 samples of sperm.

2)  Cryostorage of frozen sperm 1 month.

 

Service fees do not include:

1)  Test thaws of control embryos.

2)  Test thaws Speed Cryo frozen embryos.

3)  Test thaws of frozen sperm and IVF.

4)  Transfer of embryos into pseudopregnant recipient females. 

5)  Control of frozen germplasm. This is very important, therefore we recommend to order, and pay the small additional fees.

6)  Long term storage of embryos and sperm.

7)  Shipping charges.

 

Additional Services:

According to the customers' request, TgMM can purchase and use other mouse lines for the cryopreservation of genetically modified mice. These additional services will be charged further fees, including the extra costs of the cage(s) per diem.

 

Service fees:

See Fees for Cryopreservation. 

Rederivation of Mouse Lines

 

Background

Many thousands of different genetically engineered mouse lines have been generated over the past forty years. These new mouse lines stimulated many collaborative studies and the widespread sharing of  animals between institutions both inside the  US and internationally. This widespread distribution of mice increases significantly the possibility of transmission of different pathogens between institutions including ecto-and  endo-parasites, bacteria, mycoplasm and viruses.  Laboratory animals can become infected via horizontal or vertical transmission (Sztein et al., 2011). Horizontal transmission occurs by means of direct or indirect contact with infected animals. Many contaminants including ectoparasites (mites, lice), endoparasites (helminthes, protozoa), bacteria and many viruses are transmitted horizontally.

Vertical transmission occurs via infected gametes or penetration of a pathogen through the placenta. These pathogens are generally viruses such as Mouse Minute Virus (MVM), Lymphocytic Choriomeningitis Virus (LCMV) and hantaviruses. Maintaining pathogen-free animals is necessary to eliminate any variables that could affect the outcome of research protocols, however, there is no US or International Standard outlining what pathogens must be excluded. Because of this, importation of mice from an outside institute is often problematic since different institutions have different health standards in their animal facilities.  This problem can be resolved by rederiving contaminated mouse strains. The most popular and effective approach is to rederive mouse strains via embryo transfer.  Rederivation by embryo transfer has been shown to eliminate transmission of ecto- and endo-parasites, bacteria and mycoplasm ( Van Keuren and Saunders, 2004; Fray et al, 2008). Moreover, it has been successfully used to eliminate Mouse Hepatitis Virus (MHV), Mouse Parvovirus and other rodent viruses (Artwohl et al, 2008; Mahabir et al., 2009). Vertical transmission of viruses by contaminated sperm (for example MVM) can be eliminated by rederivation via in vitro fertilization  (Mahabir et al., 2009). The authors suggested that the presence of cumulus cells around oocytes during IVF reduced the entry of the virus into the zona pellucida and may have also absorbed some of the viruses.

To maintain specific pathogen free mice in the OHSU rodent facility, only mice from approved vendors (Jackson Lab, Charles River and Taconic) can be directly imported into colony holding rooms. Mice from other sources must undergo quarantine and shown to be free of pathogens before being released into the general population. In cases where contamination has been detected, animals must be rederived before being allowed into the general population. 

Resources and Recommendations

The Department of Comparative Medicine  has several quarantine rooms available to research staff who require the importation of animals from unapproved vendors.  If necessary, the TgMM will work with DCM to perform rederivation of "dirty" animals. Contaminated animals will be mated, 2-8 cells embryos will be subjected to a washing procedure and  transferred into pathogen-free,(SPF) foster mothers. Foster mothers will be housed in SPF isolators in special clean quarantine rooms under control of TgMM and DCM personnel. After the pups are weaned DCM personnel will collect samples  and send them to a diagnostic laboratory for evaluation. Following confirmation of pathogen-free health status, the animals will be transferred to the rodent facility for housing and use.

5 SPF ISOL 

Monitoring of rederived mice in Specific Pathogen Free Isolator

Responsible Parties and Timeline

Step

Timeframe

Protocol

Responsible Party

1

week 1

Providing of dirt mutant stud males

customer

2

week 2

Embryo generation and transfer to foster mom

TgMM

3

week 5

Biopsies collection and transfer to Charles River Lab

DCM

4

week 7

Transfer clean offspring to customer

DCM

5

variable

PCR genotype of clean offspring

customer



Service Ordering

To initiate a rederivation project, fill out and forward a Project Request Form to TgMM or contact them via email or phone.

In addition to the Project Request Form, please provide the following materials:

1)  2-3 mutant stud males from the line you are importing.

2)  2-3 virgin mutant females from the line you are importing (if you want to have homozygous mouse stock)

 

Standard rederivation service fees include:

1)  Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).

2)  Superovulation of virgin females.

3)   Collection and Transplantation of rederived embryos into pseudopregnant recipients.d) The production of guaranteed  clean animals.

4)   Animal housing charges for recipients and their offspring until weaning.

 

Service fees do not include:

1)  Animal housing charges for litters after weaning.

2)  Ear/tail tissue collection for DNA isolation for genotyping.

3)  Health testing of the foster mother.

4)  Shipping charges.

 

Additional  services:

According to customer's request TgMM can purchase and use other inbred mouse lines for the rederivation of mutant mice. Moreover, TgMM can tag, collect tail samples, extract DNA and genotype the offspring by PCR. For this purpose the animals can be housed in our colony beyond the usual 3 weeks (weaning age). These additional services will be charged additional fees including extra costs for the cage per diem.

 

Service fees:  

See Fees, Rederivation.

Molecular Genetics Service

 

Resources and Recommendations

The facility is fully equipped with tools for standard molecular genetics service, including PCR machines, centrifuges, and apparatuses for gel electrophoreses etc. Moreover, TMM is performing standard molecular genetics service such as genomic and plasmid DNA purification. Additionally, we can amplify the fragments of your genes of interest, isolate PCR products and sequence them in collaboration with the DNA Service Core.   We also have an inventory of PCR primers for the standard transgenic and reporter genes such as neomycin phosphotransferase, thymidine kinase  beta-galactosidase, green fluorescent protein and others. Moreover, TMM can design PCR primers for your newly produced animals.  To facilitate confirmation of genetic transmission of transgenes we can tag, collect tail samples, extract DNA and genotype your mice by PCR.  Additional services are possible in collaboration with the TMM.

 

Service Ordering

Please fill out, sign and submit the Project Request Form to TMM.

In addition to the Project Request Form, please provide the following materials:

1)  Your construct.

2)  A map and sequence of the plasmid.

 

Service fees:  

Contact the director TMM for individual services.