GEMouse. Added Service Guide

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Mervin Kennedy
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com Macrogen-Europe: support-europe@macrogen.

1 Contact mouse : mouse@macrogen.com Payment Inquiry: payment@macrogen.com Technical Support Macrogen Korea: support@macrogen.com Macrogen-Europe: support-europe@macrogen.com Update 68 GEMouse Added Service Guide Macrogen Korea 0F, 5 Beotkkot-ro Geumcheon-gu, Seoul 085, Rep. of Korea Tel / Fax / info@macrogen.com Copyright (C) Macrogen Inc. All rights reserved

2 Mating. Methods of Breeding Management for Strain Maintenance - Mating is performed according to the experiment purpose of TG mouse and KO mouse, strain maintenance type, and size. - Sexual maturation week of mice - males : Week 7~8, females: Week Put the male and female mice to be mated in a cage and induce mating. Males are bred separately, in principle, and it is recommended to induce mating by putting in one male mouse and five or less female mice so as not to decrease breeding efficiency. * Our company induces mating at a ratio of : or less. Once a female mouse is confirmed to be pregnant, move the female mouse to a delivery cage and prepare it for birth. When the delivery time is near, monitor the mouse twice or more a day to check if there are any abnormal symptoms including miscarriage. Once the mouse gives birth, check the condition within ~ hours. Do not replace the cage or straw litter until ~ days after delivery. A sudden change of environment at this time might give anxiety to the mother and lead her not to breastfeed or to kill the newborn mice. Pay attention to providing feed and drinks because the mother s intake of drinks and feed increases due to breastfeeding. (When drinks or feed are lacking, the mother might not raise or might prey on the newborn mice.) Perform tail-cutting for screening days after the mice are born and cut a toe to give an individual number for identification. Ablactation for mice is possible ~ days after the mice are born. Separate the female and male mice and ablactate them. / 9

3 Breeding. SPF Mouse Breeding and Strain Maintenance Recent life science research requires more precise animal testing. For such tests, it is necessary to use animals that are healthy, have clear genetic characteristics, and were produced and maintained in a breeding environment based on certain standards. However, although the general animals used in various experiments seem healthy, they are naturally infected by various microorganisms. The various in-vivo microorganisms in the animals have considerable differences in pathogenicity, and most natural infectious agents are in inactive or dormant state. Thus, there is no clinical manifestation, but they indicate pathogenicity once they are in the appropriate environmental conditions. The use of such infected animals in experiments could bring about totally different results regardless of the researcher s intention. Therefore, all infections as well as pathogenic diseases must be prevented for accurate experiments, and the specific-pathogen-free (SPF) state must be confirmed. A). SPF Maintenance System Our company has a barrier system to produce and maintain SPF mice and we operate and manage the system with Macrogen Standard Operating Procedures (SOP) for breeding management. Air Conditioning Apparatus System Our company adopts and operates high-efficiency particulate air (HEPA) filter sterilization. HEPA is a technology that filters and sterilizes the external air that enters the air conditioner with the absorption force of a blower fan through air conditioning apparatuses such as a prefilter ( um,dop 85%), medium Filter ( um, DOP 85%), heater, humidifier, or HEPA filter (0. um, DOP 99.97%) and then supplies the air to the breeding room. Air Conditioning Equipment Management System Our company is equipped with a system in which the temperature, relative humidity, air pressure, and air flow in each section of mouse cages are automatically operated, adjusted, and recorded by air conditioning hardware and software using a computer. Entry System Breeding Materials (Metal and Plastic Materials) - Breeding materials (items that are deformed by heating) are sterilized with High pressure steam sterilization (for 0~0 minutes at C) and put into a shielding facility - Breeding materials are sterilized with ethylene oxide gas and put into the shielding facility. Feed and Straw Litter - Feed and straw litter are sterilized with high pressure steam sterilization (for 0~0 minutes at C) and put into the shielding facility Breeding Animal - Breeding animals are quarantined in a quarantine room and left there Manager - The manager can access it wearing sterilized work clothes after an air-shower Disinfection of Shielding Facilities Shielding facilities are disinfected once a week using disinfectants ad UV. / 9

4 5 Breeding Environment Conditions Temperature: ± C Humidity: 50±0% Number of air changes: 5~7 times/hour Ammonia concentration: 0 ppm or less Noise: 55 db or less Illumination: 05~00 Lux B) Quality Management System Macrogen operates and manages breeding with Standard Operating Procedures (SOP) Experiment Record Management System Experiment status using mice and overall mice data including breeding are recorded, stored, and managed. Shielding Facilities Record Management System Changes in shielding facilities according to external environmental changes such as season, temperature, and weather are recorded and managed on an hourly/daily/monthly basis. Settle Plate Test A self-test for settle plate is being conducted regularly once a month. (Standard value: culture dish (three settle plates or more per blood agar) Microorganism Test Our company conducts tests four times a year by requesting them from the disease-model animal evaluation research center of the Korea Research Institute of Bioscience and Biotechnology (KRIBB), which was certified as an ICLAS Monitoring Subcenter at the th ICLAS General Assembly in May 999. C) Transgenic Mice Mating by Generation and Strain Maintenance 5 6 Founder mice are mated with normal mice to produce F. As the TG mice that our company manufacture are founder mice, each line (one line of founder mice) is mated with normal mice to produce F. It is confirmed whether germline transmission has been done in F in each line. If even one TG mouse is not observed in the screening of F mice (two to three times more with birth two or three times) obtained from the mating of founder mice, it is regarded that germline transmission is not possible. Mating and expression analysis are performed in the line where germline transmission is confirmed. Once germline transmission has been confirmed, expression analysis is performed. While an in-depth experiment is conducted after checking the expression degree, mating for line maintenance is performed. Mating is performed with the number of mice for experiment and mice size for line maintenance taken into consideration. Mating Method: TG x TG mating or TG X Normal mating are performed according to the desired gene type. If the inbreeding of transgenic mice keeps being performed, the expression of transferred genes is weakened or disappears as the generations continue. To prevent this, it is recommended to combine TG x TG mating and TG X Normal mating. Mice can be preserved by embryo freezing. When mice preservation is needed as experiments are completed and organized or as the mice preservation value is acknowledged, TG mice can be reproduced by embryo freezing of TG mice. / 9

5 D). Knockout Mice Mating by Generation and Strain Maintenance Chimera mice and heterozygous mice are produced. Chimera mice are produced by using a targeted ES clone (9/SvJ,+/-) that was obtained by electroporation. The chimera mice produced are mated with a normal mouse (+/+) of the C57BL/6N strain to produce an F mouse (heterozygous (+/-)). Germline transmission of the produced F mouse is checked by screening. The germline transmission of F mice (heterozygous founder mice) produced by the mating between chimera mice and normal mice of the C57BL/6 strain is primarily checked with mouse coat color. The coat color of F mice originating from targeted ES clones is agouti, and that of F mice originating from normal mice is black. (This applies only for F.) Germline transmission of F agouti mice is finally confirmed by screening via PCR and Southern blot. Backcross breeding is performed. Backcrossing is performed to maintain the C57BL/6N strain. Generally, as the generations from the fifth filial generation (F5) have a C57BL/6N strain of more than 96%, backcrossing is performed at least to the fifth filial generation (F5). Backcrossing should be performed over 0~ generations, in principle. * For backcrossing, heterozygous mice and normal mice (C57BL/6N) are mated. Mice Preservation by Embryo Freezing When mice preservation is needed as experiments are completed and organized or as the mice preservation value is acknowledged, KO mice can be reproduced by the embryo freezing of KO mice. * Our company provides an embryo freezing and thawing service for the convenience of customers.. SPF Mice Production and Management Many facilities and expenses are required to make the various kinds of experiment animals used in many test studies, keep them SPF and maintain and manage them, and difficult tasks such as intrauterine amputation, artificial rearing, and surrogate lactation must also be performed. Fortunately, in the case of mice as experiment animals, overall in vitro manipulation technologies from the collection of fertilized eggs via superovulation to culture, freezing, and transfer have been established for a long time and are being used stably. If such technologies are used when composing strains of SPF animals, the maintenance and management of the animals become more convenient and economical. Our company provides a service that collects fertilized eggs of mice and transfers them to an SPF surrogate mother to produce offspring, thereby establishing strains of new SPF mice. Also, we provide a service that preserves the fertilized eggs of mice for a long time using embryo freezing technology and produce and supply SPF mice when customers need them. 5 / 9

6 Embryo freezing. Embryo Freezing and Thaw The fertilized eggs of mammals can be preserved semi-permanently by being maintained at an ultralow temperature (liquid nitrogen -96 ). Such technology is widely applied to mice in particular among experiment animals, as well as livestock and humans. A) Principles and Research History of Embryo Freezing Since Whittingham et al. (97) produced the first offspring by transferring a mouse embryo that had been cryopreserved, research on embryo freezing and thawing has accelerated, and thus the freezing process has been very simplified and the survival rate after freezing and thawing has also improved. Now offspring are obtained from various mammals using the transfer of frozen embryos. The basic principle of embryo freezing is to minimize the freezing of free water in cells when an embryo is equilibrated in cryopreservation solution and soaked in liquid nitrogen for freezing, to thaw the frozen embryo at physiological temperature to recover its normal function, and to remove the cryopreservation agent by dilution after thawing. In order to meet the conditions for freezing and thawing, a lot of research has been conducted to optimize conditions such as the components and concentration of cryopreservation agents, equilibration time, temperature, and speed of freezing and thawing. For existing embryo freezing, slow freezing has usually been used. However, slow freezing requires a long time (about two hours) till the completion of freezing and an expensive program freezer to control freezing speed. Also, the method usually uses permeable cryoprotectants, and the ice crystals they generate during freezing destroy the structure of cells by applying physical pressure to cells. For this reason, Fahy et al. (98) suggested a quick-freezing technique, vitrification, to minimize the generation of ice crystals during intracellular dehydration. Vitrification has the phenomenon of hyalinzation during freezing because the viscosity of the cryoprotectants used is high. Therefore, the technique is also called hyalinzation freezing. Currently, vitrification is widely used because its manipulation is simple and the process time is short (about 5 minutes for one tube). Also, it enables freezing and thawing during the period of extensive embryogenesis from unfertilized egg to blastocyst. B) Usefulness of Embryo Freezing As scientific technologies have recently developed, transgenic mice and knock-out mice are produced and used a in a variety of research. However, maintaining each strain becomes an issue in terms of breeding management (space, efforts, and expense). To address this, the necessity of freezing the embryos of each mouse strain becomes greater. Also, due to the globalization trend, mice are actively transferred not only domestically but also internationally. Embryo freezing is playing an important role in the simplification of mice transportation and quarantine. Besides the freezing for the convenience of breeding management, embryo freezing is widely used for the purpose of production of transgenic mice, preservation of pronuclear-stage zygotes, temporary preservation of mice clean-up, and preservation of unfertilized eggs. 6 / 9

7 C). Process of Embryo Freezing and Thawing 5 Cryoprotectants Our company provides a freezing service by manufacturing DAP (M DMSO, M Acetamid, M Propylene glycol) using DMSO, which is a permeable cryoprotectant, propylene glycol, and Acetamid, which is a non-permeable cryoprotectant. Preparation of Embryo to be frozen Our company uses fertilized eggs. The fertilized eggs are collected from the oviduct of a mouse of.5dpc after mating and then a good embryo is selected for use. Freezing Process Our company provides the service with a vitrification process where the embryo is equilibrated in M of DMSO, moved to DAP for equilibration, and immersed in liquid nitrogen. Thawing Process Our company uses a thawing process where the frozen embryo is taken out of liquid nitrogen, the cryoprotectants inside the frozen embryo are eliminated by adding 0.5M sucrose, and then the embryo is moved to a culture medium. Embryo Transfer The embryo, which was confirmed to be alive, is cultured for one day and if it is developed into a blastocyst, it is transferred to a surrogate mouse mother of.5dpc. 7 / 9

8 (Mouse Ovulation and Fertilization Process) Our company s freezing and thawing process has an embryo survival rate of over 80%. Therefore, the service of our company is able to maintain strain and line even with the freezing of a small number of embryos. In particular, when patents are applied, our company satisfies the needs of customers with a rapid and stable service in the process of donation of frozen embryos. The microinjected embryo is transferred to a surrogate mouse mother, which gives birth on 9 days after transfer. After the newborn mouse grows for two weeks, its tail is cut and genomic DNA is extracted from it to check gene insertion. Protease is usually used when genomic DNA is extracted. As protease digests 8 / 9

9 thermostable polymerase which will be used later, special attention should be paid to it. Also, special care should be given to pipetting so as not to have contamination in the process of organic extraction (e.g., phenol: chloroform extraction). PCR analysis or Southern blot is performed to confirm the gene insertion in the extracted genomic DNA. Our company generally uses PCR analysis, which is accurate as well as economical in terms of both time and expense. Primer design should be specific for the trust of PCR analysis that will be used for the analysis of a founder mouse. To this end, various software can be used. But the following should be kept in mind unless there is a specific software program. Primer length should be at least 5bp, and the length between 5bp and 0bp is general. The appropriate G+C ratio of each primer is 50~60%. The consecutive sequence of Gs and Cs should be avoided in the primer sequence. The G+C ratios of forward primer and reverse primer should be similar. 5 The appropriate PCR products are 500bp~000bp. A specifically designed primer must be tested before being used in the founder mouse test. If the above is considered, PCR analysis is very reliable as a method to confirm a founder transgenic mouse and the germline transmission where its genes are transferred to offspring. 9 / 9

EMMA Cryopreservation Workshop Novel concepts in mouse production and preservation

EMMA Cryopreservation Workshop Novel concepts in mouse production and preservation Consejo Superior de Investigaciones Cientificas Madrid, Espagna May 7-8, 2012 K. C. Kent Lloyd, DVM, PhD University of