Mouse Assisted Reproductive Technology (MART) Core

• Cryopreservation of Embryos
• Cryopreservation of Sperm
• Embryo Transfer
• IVF
• Ultra-High Speed Cryo (Vitrification: embryo cryopreservation)
• Blastocyst Complementation and Microinjection (creation of genetically engineered animal models utilizing ES cell nuclear transfer)
• Post-Vitrification Morula Culture and Blastocyst Embryo Transfer
• Hybrid CRISPR/ES cell blastocyst microsurgery technology
• Manual Intra-Cytoplasmic Sperm Microinjection (Manual ICSI)

The cryopreservation of sperm and embryos is becoming increasingly more popular due to a number of advantages it has over maintaining a colony. Some of its advantages are, large financial and time savings on colony management and maintenance costs, prevention of genetic drift, and a pathogen free backup in the event of contamination.

To begin a cryogenic project at the facility, investigators are requested to provide 10 male mice of the line to be frozen and between 30-50 females of that line, or other strain desired. The number of females will be determined on an individual basis, dependent on the lines breeding record. The MART core will perform breeding to obtain the necessary animals, if mice are not available. This breeding will be an additional charge to the investigator. Following is the timeline for a cryopreservation project.

Embryo Freezing Timeline

*Each round of cryopreservation takes 5 days to 1 week. The number of rounds needed for a project will vary depending on breeding performance of the background strain. Feel free to contact us regarding to the animals needed for your specific project.

Responsibilities of the Investigator:
Provide the facility with 10 males (preferably homozygous mice). If these mice are not provided and the Facility must breed, the investigator is responsible for related charges. Animals will be transferred to MART facility protocol, but will remain under PI account for cage charges by LASC. Investigators are asked to create a backup of their line (ex. send animals to collaborators or keep one breeding cage), as there is never 100% guarantee in the cryopreservation and storage process.

Responsibilities of the Facility:
Material, time, and labor are needed for the cryopreservation of 250 or 500 embryos (to be specified by the investigator). When provided with homozygous males, the Facility will order the females (10 females per cryopreservation) and will pay the cost for housing. When it is necessary to breed for freezing, the Facility is available to do so at charge to the investigator.

Time frame

• Day 1: PMS or Hypernova injection.
• Day 3: HCG injection.
• Day 4: Perform IVF. Collect fresh sperm from sperm donor males (or thaw frozen sperm) as well as collect oocytes from hormone-primed donor females (or thaw frozen oocytes).
• Day 5: Perform embryo morphology score and embryo transfer (ET). Transfer all fertilized (2-cell) embryos into 0.5 days post coitus pseudo-pregnant females; transfer a mean of 12 embryos per infundibulum.
• Day 24: Litters are born.
• Day 45: Litters are weaned.
• Day 59: Animals are tested for pathogens and transferred to investigators.

Animals and Sperm Recovery: Investigators will need to supply the MART facility at least 1-2 male founders from which fresh sperm will be isolated and used for IVF, or with 1-2 straws of frozen sperm. Fresh sperm works better and ideally males should be 2-5 months old and proven good breeders. 2 male sperm donors tend to yield 8 to 10 straws for cryopreservation from 4 epididymides using our in-house calibrated French straws; each straw should be good for 1 IVF. MART will also provide sperm morphology, motility and density scores.

Embryo Zygosity: The MART core will cryopreserve embryos, which will be obtained from WT females or any transgenic founder females, depending on the investigator’s preferred embryo zygosity.

Disclaimer: IVF is a very tricky, capricious procedure, and its success depends on many factors. The largest determinant is strain or sub-strain used. There is great variation of success based on the strain of the animals used as well as the age and inherent reproductive performance. This variation ranges between 90% efficiency in some strains to 5% in others. The MART core, therefore, cannot guarantee successful rederivation or rescue of your line. The MART core is well experienced, and will do its best, including a second attempt, if the first round of IVF fails. The charges collected cover the IVF, the animals and per diem cage costs, as well as all supplies, time and labor. If the In Vitro Fertilization is not successful in your particular strain, there are still other options that the core can direct you to in order to save your line.

Genotyping: Since the sperm will fertilize a wild type egg, your homozygous transgenic line or knockout line will become heterozygous after IVF, needing cross breeding to achieve homozygosity.

• Embryo transfer (fresh embryos)
• Embryo transfer (frozen embryos)
• IVF (fresh sperm)
• IVF (frozen sperm)
• Caesarian section and cross-fostering
• Neonatal rederivation and cross-fostering
• Ultrahigh Speed Embryo Cryo (Vitrification)

For a complete table listing schedules and number of mice needed for rederivation visit Rederivation By Days). The MART core performs rederivations employing embryo transfer. The embryos from animals suspected of carrying pathogenic agents are removed from pathogen positive embryo donor mothers and transferred into pathogen free surrogate pseudo- pregnant mothers. Embryo transfer (ET) is the principal method for derivation of pathogen-free pups. Alternatively, pathogen-free pups may be generated using IVF as well as neonatal cross-foster rederivation. The MART core recommends this service for any line that has tested positive for pathogens, as well as any new lines being imported from other institutions to BU.

Schematic for rederivation from live mice (infected with a pathogen): Day 1: Inject hormones to donor females. Administer (PMS) or Hyperova: Day 3: Inject hormones to embryo donor females. Administer (HCG) and pair them with fertile males. Day 4: Check embryo donor females for vaginal plugs to confirm mating. Set up pathogen-free estrus females with vasectomized males. Day 5: Check estrus females for vaginal plug to confirm mating. Isolate fertilized 2-cell stage embryos from infected females, treat them with a washing medium such as FHM (Fluid Holding Medium) or M2 medium to sanitize and transfer embryos into infundibula of pathogen free 0.5-day post coitus pseudo pregnant surrogate mothers. At 19 days post infundibulum embryo transfer, possible pathogen free litters are born. Pups are weaned at 21 days old. At nearly 6 weeks old, fur and fecal pellet samples are submitted to IDEXX for PCR pathogen screening. Pathogen free pups are transferred to investigators’ pathogen free housing.

Schematic for re-derivation from frozen embryos: Day 1 – Set up clean females with vasectomized males. Day 2: Check females for vaginal plug to confirm mating. Day 3: Thaw 1-cell through morula stage embryos. Perform infundibulum embryo transfer to 0.5 days post coitus surrogate mothers. Alternatively, culture in KSOM medium to blastocyst stage. Perform blastocyst embryo transfer to 2.5 days post coitus surrogate mothers. Pathogen free litters are born at 19 days post infundibulum embryo transfer or 17 days post blastocyst embryo transfer, respectively. Pathogen free pups are weaned 21 days after birth date. At nearly 6 weeks old, fur and fecal pellet samples are submitted for PCR pathogen screening. Pathogen-free pups are transferred to investigators’ pathogen free housing.

The core personnel can assist with breeding colonies of mice generated at our school or purchased/imported. This includes mating, following the colony, application of special diet, creating a census and updating the investigator weekly. Cross breeding of mice of different genotypes and strains has been successful in the core’s hands. Embryo and tissue harvesting is also possible. In order to proceed with this service, the core personnel have to be added to the IACUC protocol of the specific project. Please contact the core to inquire about fees per colony (depending on hours of work per week).

Alternatively, creation of genetically engineered animal models may utilize gene editing (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9942237/), employing pronuclear or cytoplasmic microinjection of nucleases such TALENs and Zinc Finger Nuclease (ZNF); or CRISPR, utilizing RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA).

Another approach for creation of genetically engineered animal models involves either classical oocyte enucleation and gene targeted embryonic stem cells (ES cells) nuclear transfer or, more amenably and feasibly, ES nuclear transfer employing diploid or tetraploid blastocyst embryo microsurgery and microinjection (https://pubmed.ncbi.nlm.nih.gov/11331774/) This approach also entails blastocyst complementation, whereby the blastocyst’s endogenous gene’s niche is vacated prior to blastocyst microinjection and replaced with the foreign gene of interest carried within nuclear transfer vehicle, the ES cell nucleus, toward generation of transplant cells, tissues, or organs, for example. The BU MART facility offers the above blastocyst complementation platform.

Transgene: The injected DNA integrated into the genome of transgenic mice.

Founder line: A line of mice derived from a mouse in which the DNA was integrated in a specific site in the chromosome. From an injection of one type of DNA construct, several founder lines can be established, depending on the integration site.

Knockout mouse: a mouse in which the expression of a specific gene is ablated due to targeted insertion of interfering DNA to a gene in embryonic stem cells (by homologous recombination).

Embryonic stem cells: Cells derived from the inner cell mass of blastocysts (3-day embryos) and used for gene targeting by homologous recombination or gene editing using TALENs, Zinc Finger Nuclease, or CRISPR technologies as wells as tetraploid blastocyst embryo microsurgery (PMCID: PMC2703175). Tetraploid blastocyst ES cell nuclear transfer may also be translated toward endangered species recovery and preservation, as well as possibly toward de-extinction, underpinning overall restoration of the earth’s biodiversity, ecosystem integrity and health.

Chimeric mice: Mice developing from blastocysts injected with pre-engineered embryonic stem cells.

1. The “Forgotten Morula’’: Post-Vitrification Morula Culture and Blastocyst Embryo Transfer

MART has developed a more efficient approach to mouse line resurrection after cryopreservation, while preserving line zygosity and averting attendant lengthy congenic backcrossing. Instead of using the rest of pre-implantation stage embryos which tend to result in inefficient or capricious mouse line recovery rates after cryopreservation, this approach employs morula-stage embryo recovery, cryopreservation, and embryo culture and embryo transfer resurrection (see https://www.oxfordglobal.co.uk/wp-content/uploads/2021/04/Post-Vitrification-Morula-Culture-and-Blastocyst-Embryo-Transfer-Poster-April-22-2021.pdf).

This reproductive engineering technology offers the following recovery advantages:

• EFFICIENCY: Post-Vitrification Morulae Culture and Blastocyst Embryo Transfer may complement or improve yield versus Fresh Sperm IVF, Frozen Sperm IVF, or Laser IVF, with 100% parturition efficiency and a markedly reliable endpoint founder frequency, i.e., the number of pups generated for each rederivation project. Our pilot study had a 100% parturition rate and each experiment resulted in live pups, with a mean of 8 healthy offspring per experiment (using morulae) in comparison with 1 pup per experiment under conventional conditions (using blastocysts).

• SCALE: The above reproductive performance was achieved despite using only 30% of blastocysts created employing morula culture. As a result, the number of live pups may increase markedly with transfer of the remaining 70% of blastocysts, improving over the commonly accepted standard of at least 2 founders per experiment for transgenic mice production.

• ZYGOSITY PRE-SELECTION – ALLEVIATION OF TIME AND FINANCE CONSTRAINTS: Significantly, post-vitrification morulae culture and blastocyst embryo transfer may generate homozygous founder mice as well as a combination of choice pre-selected alleles, versus frozen or fresh sperm IVF that generate only heterozygous offspring. And the latter may require several generations of congenic back-crossing with attendant longer timelines at greater expense.

• OTHER APPLICATIONS: This technology may be useful for human-assisted reproduction as well as endangered species recovery and preservation.

2. Blastocyst Complementation for cell, tissue, and organ transplantation

MART recently advanced in vivo generation of cells, tissues, or organs with diploid blastocyst complementation in collaboration with Boston University’s Center for Regenerative Medicine toward transplant research (https://crem.bu.edu/). This tool complements in vitro methods employing organoids but may involve fewer bottlenecks due to intact genomics and attendant in vivo chemical or molecular signaling potential for differentiation and development.

Development of this tool involved adoption of tetraploid blastocyst complementation (embryo microsurgery and blastocyst microinjection – PMCID: PMC2703175) utilizing ES cells which had been cryopreserved for ten years. The resulting transgenic chimaeras were derived from transgenic Green Fluorescent Protein (GFP+) knock-in NKX-B6-01 ES cells with a 129/B6 background. Donor-derived GFP+ cells were only seen fluorescing in the airways of transgenic mice, just as hypothesized and expected.

Tetraploid blastocyst ES cell nuclear transfer may also be translated toward endangered species recovery and preservation, as well as possibly toward de-extinction, underpinning overall restoration of the earth’s biodiversity, ecosystem integrity and health.

3. Hybrid CRISPR/ES cell blastocyst microsurgery technology

This approach serves to optimize CRISPR technology (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9942237/) toward creation of non-off target CRISPR-generated mouse animal models. MART offers a hybrid CRISPR/ES cell blastocyst microsurgery technology for generation of CRISPR-generated mice on MART’s new blastocyst complementation platform (see above).The platform ideally utilizes pre-selected non-off target ES cell clones for ES cell nuclear transfer, employing diploid or tetraploid blastocyst technology as blastocyst embryo microsurgery and microinjection, averting unintended genetic variability reported in CRISPR-generated mice utilizing conventional CRISPR construct presentation into the pronucleus or cytoplasm of fertilized 1-cell mouse embryos. CRISPR has also been employed in zebra fish (https://www.nature.com/articles/s41467-022-28244-5).

4. Manual Intra-Cytoplasmic Sperm Microinjection (Manual ICSI)

Cryopreserved sperm can lose their motility after years of storage in liquid nitrogen. ICSI offers an option for “rescuing” immotile sperm that otherwise could not fertilize oocytes via conventional IVF (Fertilization and Development of Mouse Oocytes Injected with Isolated Sperm Heads1 | Biology of Reproduction | Oxford Academic (oup.com). MART’s Manual ICSI starts with generating sperm heads (sperm nuclear cells) from intact sperm (head + tail) by gentle sonication waves. It then resembles routine ES cell blastocyst microinjection, except it is carried out in 1 micro-drop of injection medium such as HTF (human tubal fluid medium), compared to conventional ICSI that requires up to 5 different injection micro-drops with different media, a technically cumbersome task even in the hands of expert embryo microsurgeons. Rather, individual, immotile sperm heads are loaded in the microinjection pipette for sperm cell nuclei microsurgical injection into oocyte oolemma in 1 microinjection micro-drops. This avoids use of the expensive ICSI piezo drill and hazardous mercury in the injection micropipette as well as the relatively challenging hurdles presented by conventional ICSI oocyte microsurgery.

Prior to breeding mice, each investigator will be trained in the use of the animal facility by ASC personnel. For detailed instructions, refer to the BUASC webpage on animal training and facility access. In addition, the staff in the MART core will be available for consultation at any time.

• Mouse Aseptic and Survival Surgery
• Embryo Culture
• Embryo Microsurgery and Microinjection – Microinjection Workstation
• Embryo Microsurgical Transfer – Infundibulum Embryo Transfer
• Embryo Microsurgical Transfer – Uterine Embryo Transfer
• In Vitro Fertilization (IVF): Frozen and Fresh Mouse Sperm
• Generation of Genetically Engineered Animal Mouse Models using DNA, mRNA, ES Cells Embryo Microinjection; Gene Editing with CRISPR or TALENs
• Microinjection for Organoid Generation
• Blastocyst Complementation toward Generation of Cells, Tissues, or Organs for Transplantation
• Embryo Microsurgical Reconstitution: Developmental Fate Assessment
• Graduate Student Practicum
• Creation of Micro-instruments for Embryo Culture, Microinjection, Embryo
• Transfer
• Estrus Selection
• Vasectomy Surgery
• Hormone Priming: Superovulation, including Hyperova Superovulation
• Generation of Embryos for Cryopreservation and Resurrection Using Embryo Culture and Embryo Transfer
• Mouse Sperm Recovery and Cryopreservation

• Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups (see also: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091892)
• Blastocyst Complementation: Comparison of Tetraploid Blastocyst Microinjection of Outbred Crl:CD1(ICR), Hybrid B6D2F1/Tac, and Inbred C57BL/6NTac Embryos for Generation of Mice Derived from Embryonic Stem Cells
• Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation
• Reproductive Engineering Manual for Lab Mouse; Center for Animal Resources and Development, Kumamoto University, Japan
• Development and Phenotypic Variability of Genetically Identical Half Mouse Mouse Embryos; Harvard Medical school, Department of Physiology and Biophysics, Lab for Human Reproduction and Reproductive Biology; article publication with Development, Cambridge and Oxford universities, England.
• Post-Vitrification Morula Culture and Blastocyst Embryo Transfer Complements Conventional Assisted Reproductive Technologies: an example of recent MART research and Development
• Laser IVF and Intracytoplasmic Sperm Injection: Embryo Microsurgery
• Preparation of Embryonic Stem Cells for Blastocyst Microinjection and Complementation