Ming, the experiment needs a large batch of offspring that are close in age. Is there a way to obtain them quickly?

 

In this case, we can consider rapid colony expansion via IVF. Let's discuss IVF.

 

IVF for Rapid Colony Expansion
 

IVF (In Vitro Fertilization) for rapid expansion utilizes the sperm from 1-2 male mice with the correct genotype, combined with oocytes from superovulated female mice, to form embryos. These embryos are then transferred to pseudopregnant recipient females, ultimately yielding a large number of age-synchronized offspring.

Challenges associated with traditional natural mouse breeding, such as long breeding cycles, low expansion efficiency, and difficulty in pathogen rederivation, can be addressed through IVF.
 

Characteristics of IVF
 

Advantages:
 

●Rapid, Large-Scale Production:  1-2 genotype-confirmed male mice can produce at least 50 or more offspring within approximately 45 days. Offspring have consistent birth dates, typically within a 2-3 day window, greatly reducing experimental variability.

●Resource Conservation:  For male-sterile, aged, or endangered strains, IVF technology allows extraction of sperm for in vitro fertilization, ensuring the continuation of valuable genetic resources.

●Quality Assurance:  Pseudopregnant recipient (surrogate) females are SPF-grade, guaranteeing the health status of the resulting offspring.

Disadvantages:

●Best suited for scenarios  where the egg donors are readily available wild-type females (e.g., C57BL/6J).

●Most efficient for producing heterozygous offspring.  If homozygous mice are required, subsequent breeding or another round of IVF is necessary.
 

Core Steps of the IVF Procedure
 

1. Superovulation and Oocyte Collection

●Hormone Treatment:  Select 3-4 week-old female mice. Inject PMSG (10 IU per mouse) at 16:30, followed by hCG (10.0 IU per mouse) 47 hours later.

●Oocyte Retrieval:  Euthanize females 16 hours post-hCG injection. Isolate the oviducts. Flush the ampullae with M2 medium to collect cumulus-oocyte complexes (COCs). Transfer COCs to drops of HTF medium.
 

2. Sperm Collection and Capacitation

●Male Preparation:  Select healthy male mice aged 8-24 weeks. Euthanize by cervical dislocation and quickly excise the cauda epididymides. Place them in IVF medium containing 5% Serum Protein Substitute (SPS).

●Sperm Capacitation:  Release sperm by gently puncturing the epididymal tissue. Incubate at 37°C for 1 hour. Sperm swimming to the edge of the medium droplet indicates successful capacitation.
 

3. In Vitro Fertilization

●Gamete Co-incubation:  Transfer COCs to fertilization drops (containing HTF medium). Add 5-15 µL of capacitated sperm per 10 oocytes. Incubate for 6-7 hours at 37°C in a 5% CO₂ incubator.

●Fertilization Assessment:  Observe presumptive zygotes the next day. Confirm pronuclear formation or development to the 2-cell stage. Calculate the fertilization rate (Number of 2-cell embryos / Total number of oocytes).
 

4. Embryo Transfer

●Pseudopregnant Recipient Preparation:  In advance, mate vasectomized males with normal females to induce pseudopregnancy. Select recipients at 0.5 days post coitum (dpc) for oviduct transfer or 2.5 dpc for uterine transfer.

●Transfer Procedure:  Under a stereomicroscope, transfer 10-15 two-cell embryos into the recipient via the infundibulum or a small incision in the oviduct/uterine wall. Post-operative care includes maintaining body temperature and administering antibiotics to prevent infection.

●Pregnancy Monitoring:  Monitor recipients for parturition 19-21 days post-transfer. Wean pups at 21 days for experimental use.

 

References:

[1] Tsinghua University - Laboratory Animal Research Center

[2] Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)

[3] Relevant technical patent resource.

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