Embryo Transfer and Purification Platform

For more detailed information, please contact MS.Gao saifeigao@hkust-gz.edu.cn or Ms.Li aerinyaruili@hkust-gz.edu.cn.

What is the purpose of biological purification？

To reduce or prevent pathogens from being transmitted through live mice to other mice within barrier facilities, thus causing contamination of animals inside the barrier. LAF requires that genetically engineered mice undergo biological purification before entering the breeding area.

How are purification methods selected?

Embryo transfer is a common method of biological purification. Embryo transfer involves isolating pre-implantation embryos from the oviduct or uterus and transferring them to the oviduct or uterus of pseudopregnant recipient mouse. The transferred embryos can implant, develop, and be born within the pseudopregnant mouse.

There are two methods for obtaining embryos: (1) natural mating (in vivo purification): male mice are mated with superovulated female mice, following which the presence of a copulatory plug is checked. The female mice are then euthanized after plug observation, and the fertilized eggs are collected for embryo transfer. This method is commonly referred to as in vivo purification; (2) in vitro fertilization (IVF): this involves collecting sperm and eggs, performing in vitro fertilization (IVF), and then conducting embryo transfer. This method is usually referred to as IVF purification.

The success of this process is influenced by numerous factors, such as the quality of the sperm, which directly affects the fertilization rate. Although our extensive experimental data indicate that the fertilization rate is generally over 60%, we cannot guarantee a successful outcome on the first attempt.

IVF or Natural mating, how to choose？

The choice of IVF and in vivo purification is mainly based on whether male and female mice can mate effectively. In either purification method, the number of pups born is directly related to the number of female mice providing embryo. If the rate of suppository is high, the number of births obtained by IVF purification and in vivo purification is not different.

1. IVF Purification: This method requires 1-2 male mice, which are euthanized to collect sperm. All superovulated female mice are also euthanized. This approach does not necessitate monitoring for copulatory plug presence, effectively avoiding delays caused by failed mating between male and female mice that could hinder purification. It is generally recommended to use more than two male mice, and if they can be mated with wild-type female mice such as B6/J for purification, IVF purification should be directly employed.
2. Natural Mating Purification: If the number of male mice is insufficient (fewer than two) or if the male mice need to be retained for further use after mating, and they cannot be mated with wild-type female mice such as B6/J for purification (due to homozygosity, polygenic factors, or other reasons), the natural mating method should be prioritized.

How to apply for purification service?

To apply for purification services, please download and complete the document titled "E001–Purification Service Application Form - V1". The form can be downloaded from the "Form Download" section, which contains detailed instructions. Please read and fill out the relevant information carefully.

What is the service duration？

Different strains of laboratory animals exhibit significant variations in ovulation, conception, and other related procedures; therefore, the time required for purification may vary. Generally, offspring can be obtained at four weeks of age approximately 8-10 weeks after the purification process begins.

How is the quality of the purified mice ensured to meet SPF standards? Is testing necessary?
LAF has a comprehensive sentinel mouse and random sampling system to ensure that the quality of the purified animals meets the national standards for SPF classification.

For more detailed information, please contact MS.Gao saifeigao@hkust-gz.edu.cn or Ms.Li aerinyaruili@hkust-gz.edu.cn.

I. Requirements for age and quantity of male mice: They should be over 8 weeks old, preferably not exceeding 8 months. Additionally, the health status of the mice should be considered, ensuring that they can be introduced to female mice for mating.

a) For single-gene modified mice, such as Tg/KO/KI, if they are heterozygous and can be mated with common strains like B6 for purification, please provide at least 2 male mice. If there are 3 male mice available, one positive male mouse can be considered for culling (if there are 3 male mice, one can be culled), and sperm can be collected for IVF with B6, which can yield a larger number of offspring at once.

b) For single-gene modified mice, such as Tg/KO/KI, if they are homozygous and cannot be mated with B6 for purification, the research team should provide male and female mice for purification as required, generally in a 1:1 or 1:2 ratio. It is recommended to expand the population to ensure an adequate number of female mice.

c) For multi-gene modified mice, such as TKO/DKO/cKO, breeding must be conducted first to obtain offspring before mating or IVF purification. If they are homozygous, please provide at least 2 male mice, with the male-to-female ratio provided as 1:1 or 1:2.

II. Requirements for age and quantity of female mice: Female mice need to undergo hormonal superovulation, and are generally required to be between 3-5 or 9-12 weeks old, avoiding the hormone-insensitive period of 6-8 weeks. Additionally, the health status of the mice should be considered, ensuring that they can exhibit copulation for ovulation.

The quantity of female mice should be determined based on the purification method and the anticipated number of offspring.

Note: In purification experiments, all female mice that exhibit copulation will be euthanized to collect embryos; however, not every female mouse that exhibits copulation will yield normally developed embryos. Therefore, it is essential to retain a sufficient number of female mice for breeding to avoid the risk of losing the breeding stock. 

For more detailed information, please contact MS.Gao saifeigao@hkust-gz.edu.cn or Ms.Li aerinyaruili@hkust-gz.edu.cn.

I. From the perspective of thawing:

1. The offspring obtained after thawing cryopreserved embryos will have the genotype of the frozen mouse strain.
2. For sperm cryopreservation, thawing requires in vitro fertilization (IVF) to produce offspring, and it is necessary to consider the egg donor. If the donor is B6/J, then the offspring obtained after thawing will be heterozygous, resulting from mating with B6.

II. From the perspective of cryopreservation costs:

1. Embryo cryopreservation requires a significant amount of time during the freezing process, primarily due to the cycle needed to obtain a certain quantity of embryos. The freezing costs are high, but the thawing process is simple and inexpensive.
2. In contrast, sperm cryopreservation is relatively inexpensive and quick, but thawing requires IVF, which incurs higher costs. Additionally, thawing sperm can yield many offspring at once.

III.How to choose which preservation method to use?

1. For multi-gene knockout, embryo cryopreservation is generally recommended.
2. For single-gene knockout or transgenic mice, sperm cryopreservation is recommended.
3. Each situation should be assessed individually. The specific choice depends on the number of existing animals.

IV. What are the costs for embryo cryopreservation, sperm cryopreservation, and thawing?

Please refer to the "Fee Standards."

For more detailed information, please contact MS.Gao saifeigao@hkust-gz.edu.cn or Ms.Li aerinyaruili@hkust-gz.edu.cn.

I. What is rapid mass expansion?

Rapid expansion refers to a method of obtaining a large number of offspring mice using 1-2 male mice through in vitro fertilization (IVF).

II. What is the principle of rapid mass expansion?

The core of rapid expansion is the use of IVF technology, where capacitated sperm is combined with oocytes for fertilization in vitro, followed by implantation into pseudopregnant female mice to obtain offspring.

III. What are the advantages of rapid mass expansion?

 •1-2 positive male mice can produce at least 50 or more offspring within 45 days.

• Offspring are born around the same time, generally within a 2-3 day range.

IV. What are the disadvantages of rapid mass expansion?

• It is more suitable when the embryo donor is an easily obtainable female wild-type mouse, such as B6/J.

• It is more suitable for producing heterozygous offspring. If homozygous offspring are needed, additional mating or IVF is required.

For more detailed information, please contact MS.Gao saifeigao@hkust-gz.edu.cn or Ms.Li aerinyaruili@hkust-gz.edu.cn.

In the event of sudden death of important strain male animals or if a male is unable to successfully mate with a female, we can attempt to address this issue through in vitro fertilization (IVF) experiments. Generally, strains can be attempted for IVF within a few hours after the male mouse has died, the sooner, the better (promptly collect and preserve the epididymis of the deceased male mouse).

In vitro fertilization (IVF) involves mixing capacitated sperm with mature oocytes in an external environment to complete the fertilization process.

Key considerations for IVF include:

1. Zona Pellucida Hardening: Over time, it becomes increasingly difficult for sperm to penetrate the zona pellucida. The longer the oocyte remains outside the body, the more hardened the zona pellucida becomes. It is crucial to collect oocytes quickly.
2. Temperature is Critical: All instruments and materials used should be maintained at 37°C whenever possible. Setting up a small incubator next to the microscope can help minimize the exposure of the fertilization dish to room temperature and air.
3. pH Level is Important: In the presence of air, the pH level of the culture medium can rise rapidly. It is advisable to keep the fertilization droplet within the incubator.
4. Volatile Organic Compounds in the Laboratory: These can significantly impact the success of IVF and the developmental capability of embryos after transfer. The laboratory environment should be maintained in optimal air quality conditions.

For more detailed information, please contact MS.Gao saifeigao@hkust-gz.edu.cn or Ms.Li aerinyaruili@hkust-gz.edu.cn.