User guidelines for KCTT Transgenic Core Facility

Which technique to choose depends mainly on the aim and type of modification needed.

• Transgenic mice: Using this technique, one typically aims to create a mouse where a gene is (over)expressed. This is achieved by microinjection of a linear DNA construct into the pronucleus of mouse zygotes. The microinjected zygotes are then implanted by a surgical procedure into pseudopregnant surrogate female mice. By choice of heterologous promoter to drive the transgene, one could control its spatiotemporal expression in the mouse.

  This type of model is normally the quickest and least expensive to produce. However, there are some putative limitations with this type of model to be aware of. One is that you normally cannot control how many copies and where in the genome the transgene will integrate since this is in essence a random process. Consequently, this often will result in transgene-positive, first-generation (G₀) mice having different inter-individual levels of expression. For that reason, each G₀ transgenic mouse should be regarded as unique and be the potential founder of an independent transgenic line. 

  As a golden rule, if possible, one should aim to generate and characterize several independent lines (i.e. generating several G₀ founders). We also recommend testing the expression of the transgenic construct in a suitable cell line before embarking on the transgenic mouse generation step. C57BL6/N and C57BL6/J are offered as standard strain backgrounds for the transgene. We can handle standard plasmid-based constructs as well as PACs and BACs. We can perform linearization, agarose gel isolation, and purification of your transgenic construct.

   

• Gene targeted mice: Using this technique, one can create knockouts, knockins, conditional alleles, etc., by introducing a targeting vector via electroporation into embryonic stem cells (ESCs). The aim is for the vector to target and modify the endogenous gene using the cell-intrinsic process of homologous recombination. The resulting gene targeted ESCs are then used for making chimeric mice via microinjection into mouse blastocysts. The manipulated blastocysts are then implanted by a surgical procedure into pseudopregnant surrogate females. A gene targeting project is divided into two main parts, which is also reflected in the two iLab service request forms: 1) gene targeting in ESCs and 2) blastocyst injection of targeted ESC clones. We have different ESC lines (strain background in parenthesis) available for targeting: E14 (129/OlaHsd), RW-4 (129X1/SvJ), R1 (129X1/SvJ × 129S1/Sv), JM8A3 (C57BL6/N-A^tm1Brd).

  We can give advice on building the targeting vector but currently do not offer the service of building it. However, we can linearize and purify the vector for the ESC electroporation. For the positive ESC clones surviving the selection, we can offer to do the primary screening, or we can send you the crude ESC lysates for own screening. Positive candidate ESC clones from the primary screening will be thawed, expanded, and be subjected to confirmation by Southern blot hybridization and determination of ploidy status. Chimeric mice will subsequently be generated from the validated ESC clones.

  Several international repositories, summarized at the homepage of the International Knockout Mouse Consortium (IKMC), have already targeted ESC clones available for purchase, and we can perform blastocyst injection of such imported ready-made clones to generate chimeras as well. The produced chimeras can either be sent to you for breeding for germline transmission or we can perform this step for you.

   

• Gene edited mice: For creating knockouts and smaller knockins (<3 kb), this is normally the method of choice since it is typically faster than the ESC-based gene targeting technique mentioned above. Using the technique for making mice with longer knockins than ~3 kb and conditional knockouts might also be possible although here ESC-based targeting can start to be competitive.

  The method involves electroporation (standard for INDEL mutations and knockins with repair templates <200 nucleotides) or pronuclear microinjection (standard for knockins using repair template >200 nucleotides) of mouse zygotes with CRISPR/Cas components. The manipulated embryos are then implanted by a surgical procedure into pseudopregnant surrogate female mice. Various Cas proteins such as Cas9, Cas9 nickase, Cas12a (Cfp1), are available for different approaches. C57BL6/N and C57BL6/J are offered as standard strain backgrounds for the editing.

  For help in designing the editing strategy and getting the optimal CRISPR components, we collaborate with the CRISPR Functional Genomics (CFG) unit at KI. One thing to be aware of with the CRISPR/Cas technique is that the generated G₀ offspring are often mosaics and requires breeding one generation to ensure a homogenous and heterozygous G₁ founder animal. In addition, another issue is that sometimes unwanted on- and off-target genetic artefacts might accompany the desired edit. Therefore, sequencing over relevant areas and other genetic quality controls is strongly recommended to confirm the integrity of the edited allele. We can offer to perform both the breeding to obtain G1 animals and the necessary genetic quality controls.

For an excised and purified transgenic construct without the vector backbone, we would like to receive at least 2-3 µg, preferably more, in a concentration of at least 10 ng/µl in microinjection buffer [10 mM Tris-HCl, 0.2 mM EDTA, pH 7.4]. We can provide you with the microinjection buffer. We will then process it further and make the necessary dilution suitable for the microinjection.

If supplying the transgenic construct as a circular plasmid for the service of letting us perform the excision and purification of the transgenic DNA fragment, we would like to receive at least 25-30 µg or more. Please also supply a plasmid map and photo of a diagnostic agarose gel run of a restriction enzyme digest with the relevant enzyme(s) that is used to liberate the transgenic construct from the vector backbone.

For a linearized gene targeting vector or circular vector for linearization by us, we would like to receive at least 30 or 60 µg, respectively, at ~1 µg/µl in 10 mM Tris-HCl, pH 7.4-8.0. Please also supply a plasmid map and photo of a diagnostic agarose gel run of a restriction enzyme digest with the relevant enzyme(s) that is used to linearize the targeting vector (for gene targeting in ESCs, it is not necessary to remove the plasmid vector backbone).

The transfers of the manipulated pre-implantation embryos are conducted into FELASA SOPF quality surrogate females in a high safety barrier with restricted access by authorized personnel only. In addition to the routine health monitoring of the barrier itself according to the FELASA guidelines, each specific new strain’s surrogate females used in the embryo transfer are health screened before release of their offspring to the customer. The standard health screen is included in the service price. If additional tests beyond the standard are required by the receiving animal facility, it is possible but extra charges apply.

Any cost(s) for housing after weaning the G₀ offspring and beyond and sending of ear biopsies will be charged by the KM-W animal facility separate from the KCTT charges. Likewise, the purchase cost of any wild-type animals used for the optional breeding of the G₀ offspring will normally be billed directly from the animal vendor.

For shipping the produced mice, in addition to the direct shipping cost from the courier company, additional administrative costs may accrue for handling paperwork, coordinating logistics, and any necessary local or district veterinary services, all depending on the requirements set by the place of destination.

The recommended general guidelines for typical strains in terms of number and age of mice and/or cryopreserved material needed for the rederivation to be successful are shown below. However, some atypical strains may have other requirements.

• For rederivation from frozen sperm:
  • We recommend obtaining at least 2 cryo straws of sperm from the supplier.
  • 3-6 females in the ages 3-4 and/or 8-12 weeks.
    1. The females will be superovulated and sacrificed for the procedure and are normally wild-type ordered by us directly from approved vendors.
• For rederivation from frozen embryos:
  • We recommend obtaining at least 2 straws with a minimum total of ~60 embryos (preferably 2-cell stage) or more from the supplier.
• For rederivation from live animals via IVF:
  • 2-3 males in the age of 3-8 months.
    1. The males should have been proven fertile, the more recent the better.
    2. If a male is taken from an ongoing breeding, it should be separated from the female at least 5 days prior to the procedure to ensure sufficient sperm count recovery.
    3. The males will be sacrificed for the procedure.
  • 3-6 females in the ages 3-4 and/or 8-12 weeks.
    1. The females will be superovulated and sacrificed for the procedure and are normally wild-type ordered by us directly from approved vendors but use of own females is often possible if meeting the age requirements.
• For rederivation from live animals via natural matings:
  • 5-10 males in the age of 3-8 months.
    1. The males do not need to be sacrificed for the procedure.
  • Equal number of females as males (mating 1:1) in the ages 3-4 and/or 8-12 weeks.
    1. The females will be superovulated and sacrificed for the procedure.
    2. More females may be needed for another round of matings to achieve enough embryos.

The transfers of the pre-implantation embryos are conducted into FELASA SOPF quality surrogate females in a high safety barrier with restricted access by authorized personnel only. In addition to the routine health monitoring of the barrier itself according to the FELASA guidelines, each specific new strain’s surrogate females used in the embryo transfer are health screened before release of their offspring to the customer. The standard health screen is included in the service price. If additional tests beyond the standard are required by the receiving animal facility, it is possible but extra charges apply.

We strongly recommend genotyping the rederived mice to ensure that you have obtained the expected strain. Finding out much later that you have received the wrong strain could be very costly both in terms of money and time lost. As standard included in the service, ear punch biopsies for genotyping are provided from the rederived mice.

Any cost(s) for housing after weaning the offspring and beyond and sending of any ear biopsies will be charged by the KM-W animal facility separate from the KCTT charges. Likewise, the purchase cost of any wild-type egg donor females will normally be billed directly from the animal vendor.

For shipping the produced mice, in addition to the direct shipping cost from the courier company, additional administrative costs may accrue for handling paperwork, coordinating logistics, and any necessary local or district veterinary services, all depending on the requirements set by the place of destination.

Which cryo-format to choose depends on the needs and finances. In terms of long-term recoverability, there is no apparent difference between the two formats. Sperm cryopreservation is less expensive, however, only a haploid genome is preserved. This means that to rederive the strain, one will need to perform IVF and for that will need eggs. 

If the original strain is no longer available at the time of a hypothetical future rederivation, this typically means that wild-type females of best matching strain background will be used as the egg donors. One consequence is that any homozygosity of gm alleles present in the parental strain will not be recovered in the first-generation offspring and one will need to re-establish it by heterozygous intercrossing.

If many homozygous alleles are present, this is clearly a rather time-consuming exercise. Also, if the genetic background of the original strain is unknown and/or unique, introduction of a new strain’s haploid genome from the egg donors might, in the worst case, not fully recapitulate the original strain’s phenotype. For strains with such properties, one should probably consider cryopreserving embryos rather than sperm.

The recommended general guidelines for typical strains in terms of number and age of mice needed for the cryopreservation to be successful are shown below. However, some atypical strains may have other requirements.

• For sperm cryopreservation:
  • 2-3 males in the age of 3-8 months.
    1. The males should have been proven fertile, the more recent the better.
    2. If a male is taken from an ongoing breeding, it should be separated from the female at least 5 days prior to the procedure to ensure sufficient sperm count recovery.
    3. The males will be sacrificed for the procedure.
• For embryo cryopreservation via IVF:
  • Males according to the criteria specified for sperm cryopreservation above.
  • 8-10 females in the ages 3-4 and/or 8-12 weeks.
    1. The females will be superovulated and sacrificed for the procedure.
• For embryo cryopreservation via natural matings:
  • 10-15 males in the age of 3-8 months.
    1. The males do not need to be sacrificed for the procedure.
  • Equal number of females as males (mating 1:1) in the ages 3-4 and/or 8-12 weeks.
    1. The females will be superovulated and sacrificed for the procedure.
    2. More females may be needed for another round of matings to achieve enough embryos.

For sperm, we normally cryopreserve 10 individual straws where one of them is used for quality control. One straw is normally sufficient to rederive the strain in one experiment.

For embryos, we normally cryopreserve 250-500 embryos divided into 10 or more individual straws where one of them is used for quality control. One to two straws with a total of about 40-70 embryos is normally sufficient to rederive the strain in one experiment.

One factor to consider that may influence how many straws should be cryopreserved is the strains characteristics, particularly those concerning reproductive parameters. If the strain for example has reduced gamete functionality or embryo viability, one may want to increase the numbers frozen. Also, the participating sperm or egg donors’ zygosity status have an impact on the numbers; less is generally needed if all donating animals are homozygous for the desired gm allele(s). A third aspect to factor in is how often one anticipates to rederive the strain in the future.

Under normal circumstances, if having the recommended number and age of animals at hand, we complete the cryopreservation request of a strain in one experimental session. However, if the participating animals’ sperm or egg quality is poor for example, additional sessions might be needed to reach the recommended quantity. Also, for embryo freezing using natural matings, often more than one session is needed to reach enough embryos. Any extra sessions are offered at 50% of the list price.

For the standard sperm quality control (QC), we thaw one straw and assess the sperm motility and general appearance. If requested, we can perform extended or premium QC. For the extended QC, we make a functional test of the thawed sperms by performing IVF and assess the fertilization rate as well as culture the resulting embryos in vitro and quantify how many develop to blastocysts. For the premium QC, we make a transfer of the IVF-generated embryos into surrogate females and assess the number of pups born.

For the standard embryo QC, we thaw one straw and culture the resulting embryos in vitro and quantify how many develop to blastocysts. For the premium embryo QC, after thawing we also make embryo transfer and record the number of born pups.

The embryos and sperm are frozen and kept in designated cryo straws, which are stored in cryo cassettes kept in liquid nitrogen tanks. Half the number of straws of a strain are stored at two different locations each to increase safety. This mirroring of the cryobank minimizes the risk of losing the strain should there be a catastrophic event at any of the two sites. There is an annual cryostorage fee per strain to cover maintenance and administrative costs.

We are part of the European Mouse Mutant Archive (EMMA) network, which is the primary mouse strain repository in Europe. If you are the creator of a GMM strain that you think could be of value for the scientific community, you may be able to deposit it in the EMMA archive through a free of charge cryopreservation. Find out more about what are the requirements, how the strain is evaluated for acceptance, intellectual property rights, and the online submission form.

Any cost(s) for purchasing and housing of the egg and/or sperm donor animals, and/or animal transport are not included in the standard service price and will be charged separately.