Is your gene-edited mouse breeding inefficient, with low litter sizes, low survival rates, or even sterility?
 

Don't worry! Difficulties in breeding gene-edited mice are often related to the following common issues. Let's explore them.
 

External Causes
 

1. Selecting Breeder Mice

1.Females:  Should be selected at 6 weeks of age. The sexual maturity period for female mice is 6-8 weeks, with the optimal breeding period between 2-6 months of age. It is important to check females for conditions like double vagina, absence of vagina, vaginal septum, or vaginal atresia.

2.Males:  Sexual maturity occurs slightly later in males. Select males at 8 weeks of age as breeding studs. Check for issues like absence of penis, absence of testes, or genital injuries. To increase conception rates, the male-to-female ratio for pairing can be 1:2 to 1:3.

2. Environmental Factors

1.Light and Noise:  Place cages in a quiet, dimly lit area to provide an ideal, comfortable living environment for the mice.

2.Housing Conditions:  Control the ambient temperature to 20-26°C and humidity to 40-70%. Provide soft, warm bedding (such as cotton or wood shavings) to ensure females can nest comfortably, thereby improving breeding success.

3. Nutritional Factors

1.Supplement Diet:  Add vitamin E-rich foods, such as sunflower seeds, to support healthy fetal development.

 

Internal Issues Caused by Genetic Editing
 

1.Reproductive System Impairment:  The gene editing process may affect genes related to reproductive organ development, gamete formation, sex hormone synthesis, or mating behavior, leading to sterility or severely reduced fertility. For example, in the Prm1-Cre  mouse strain, Cre recombinase is active during spermatogenesis. However, when Prm1-Cre  is bred with certain floxed mice, male offspring may become sterile.

2.Placental Defects, Umbilical Cord Malformations:  Homozygous knockout of certain genes (e.g., Gjb2) can cause placental defects leading to embryonic death. Furthermore, some genotypes (e.g., homozygous knockouts) may cause embryonic lethality, making it impossible to obtain homozygous offspring.

3.Homozygous Sterility:  Some genes (e.g., Kras, Bmp7), when knocked out homozygously, allow mice to survive but result in abnormal reproductive system development, preventing natural breeding.

4.Imbalanced Offspring Sex Ratio:  Editing genes related to sex chromosomes or sex determination can affect embryonic sex differentiation, leading to a significantly reduced number of offspring of one sex, deviating from the normal sex ratio.

5.Reduced Litter Size:  For example, FMR1  knockout mice have significantly smaller litter sizes compared to wild-type (WT) mice. The absence of the FMR1 gene directly leads to reduced fertility in mice.
 

So, how can we solve or avoid the problem of poor breeding efficiency in gene-edited mice?
 

Next, we introduce a technology that can circumvent these issues – the TurboMice™ Tetraploid Complementation Technology.

 

Tetraploid complementation technology was first proposed in 1990 and applied to study the totipotency of stem cells. Between 2001 and 2015, this technology saw further development and application. However, the birth rate of mice produced via traditional tetraploid complementation was low, at most 1%-5%. MingCeler Biotech  has developed a new generation of model mouse technology – TurboMice™ Tetraploid Complementation Technology. This technology combines tetraploid complementation with precise gene editing and mouse embryonic stem cell (mESC) technologies. By establishing and optimizing the engineering system, the birth rate of mice has been significantly increased to 30%-60%.
 

The specific principle is as follows:  Normal mouse 2-cell stage embryos (diploid) can be fused via electrical pulses to form tetraploid embryos. Tetraploid embryos have developmental defects and can only form extra-embryonic tissues, such as the placenta and umbilical cord. Embryonic stem cells can differentiate into all cell types within the embryo. However, due to limitations in spontaneous differentiation ability, embryonic stem cells cannot form the placenta.
 

By aggregating embryonic stem cells with tetraploid embryos, a new reconstructed embryo is formed. In this embryo, the tetraploid cells contribute onlyto extra-embryonic tissues (like the placenta) and not to the embryo proper. The embryo proper develops entirelyfrom the diploid embryonic stem cells, which can develop into a complete mouse individual.
 

In simpler terms, TurboMice™ Tetraploid Complementation Technology  is "mice from cells." It involves directly gene-editing stem cells, which then develop directly into mice, allowing for the generation of target homozygous mice in a cycle of 2-4 months.
 

Note: Low breeding efficiency with Cre driver mice?  TurboMice™ Tetraploid Complementation Technology bypasses traditional breeding steps. The founder mice are 100% the target genotype, allowing you to obtain the experimental target mice (Cre-positive, floxed homozygous) in one step.
 

Traditional technologies  like pronuclear microinjection and ES cell targeting chimera generation adopt a "mice from mice" model, requiring 2-3 generations of breeding and screening to obtain homozygous mice. TurboMice™ Tetraploid Complementation Technology  bypasses the time-consuming breeding and screening steps of conventional methods. It avoids issues like reproductive impairment, homozygous sterility, imbalanced sex ratios, and small litter sizes. It enables the direct, batch preparation of gene-edited target homozygous mice from mouse embryonic stem cells, ensuring a stable supply.

TurboMice™ Tetraploid Complementation Technology  can also compensate for breeding difficulties caused by placental defects, umbilical cord malformations, etc., fundamentally improving the birth rate of mice.

Choose our custom gene-editing mouse service, and receive 2 years of complimentary embryo cryopreservation.  For future mouse needs, simply notify us two months in advance, and we will deliver live mice in batches, ensuring the long-term stable supply of high-value, complex models and continuity of your experiments. We welcome your inquiries!

 

MingCeler Biotech Animal Center
 

The MingCeler Experimental Animal Research Center operates using a barrier system combined with IVC (Individually Ventilated Cages). The barrier area includes quarantine rooms, clean supply receiving rooms, clean supply storage rooms, surgical rooms, and three independent housing rooms. Laminar flow hoods are installed in the quarantine rooms, surgical rooms, and all three housing rooms to ensure sterile operating conditions. Regarding biosafety control, personnel access, material access, animal handling procedures, facility and equipment management, and early warning mechanisms are all governed by established operating procedures, with training and strict assessments implemented. Most non-autoclavable items are sterilized via irradiation at specialized companies. An annual animal quality monitoring plan has been established, clearly defining the conditions for animal access.

 

References:

[1] Dai P, Ma C, Chen C, Liang M, Dong S, Chen H, Zhang X. Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines. Biomolecules. 2024 Apr 28;14(5):529. doi: 10.3390/biom14050529. PMID: 38785936; PMCID: PMC11117649.

[2] Li Q, Cui C, Liao R, Yin X, Wang D, Cheng Y, Huang B, Wang L, Yan M, Zhou J, Zhao J, Tang W, Wang Y, Wang X, Lv J, Li J, Li H, Shu Y. The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice. Cell Mol Life Sci. 2023 May 13;80(6):148. doi: 10.1007/s00018-023-04794-9. PMID: 37178259; PMCID: PMC10182940.

[3] Fan HY, Shimada M, Liu Z, Cahill N, Noma N, Wu Y, Gossen J, Richards JS. Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation. Development. 2008 Jun;135(12):2127-37. doi: 10.1242/dev.020560. PMID: 18506027; PMCID: PMC3541831.

[4] Xiao Guohong, Ye Qiuxian, Yang Jie, Chen Shengqiang, Sun Weiwen, Huang Xiaohong, Gan Ting. Effect of FMR1 gene knockout on reproductive function in female mice. China Journal of Modern Medicine, 2014, 24(4): 7-11.

[5] Nagy A, Gócza E, Diaz EM, Prideaux VR, Iványi E, Markkula M, Rossant J. Embryonic stem cells alone are able to support fetal development in the mouse. Development. 1990 Nov;110(3):815-21. doi: 10.1242/dev.110.3.815. PMID: 2088722.

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