冷冻保存和辅助生殖
The generation of genetically engineered mice is costly and time-consuming. Because of this investment, it would be wise to bank embryos of a valuable mouse strain to guard against the possibility of losing the line in case of an infection or disaster. In addition, at the end of a project, it is often desirable to maintain a line in the eventuality that it will be required in the future. In this instance, it can be costly to maintain the line as well as take up valuable animal space. To accommodate investigators in either of these instances, the Genetically Engineered Mouse Facility (GEMF) offers mouse cryopreservation services.
胚胎冷冻
200年我们将同行都有异性的胚胎zygous males mated to wild-type females (for those strains amenable to the procedure) or 100 2-cell embryos for homozygous males mated to wild-type or homozygous females. Approximately 200 or 100 two-cell embryos or compacted morula (depending on zygosity) from the mouse line of interest will be collected by the GEMF staff, cryopreserved and stored in liquid nitrogen. A minimum of 10 males that are between 8 weeks and 6 months of age are required. If the investigator wants to freeze homozygotes, females for superovulation will need to be provided. For C57BL/6 and 129 strains, females should be 21-25 days of age at use. We generally average 15 good embryos from a C57BL/6 or hybrid, non-transgenic, superovulated female that is mated to a fertile male. If mice can be frozen as heterozygotes, the GEMF will obtain non-transgenic females of the correct age. The GEMF will superovulate the females for both homozygous and heterozygous strains. To verify the success of the procedure, one straw per freeze will be thawed, and the embryos cultured to the blastocyst stage. If the mouse line is unable to be cryopreserved as embryos, we will only freeze sperm from the line. While we are generally very successful at producing animals from frozen sperm, there are outside sources that we can recommend for recovery of the line from inactive sperm, if this proves necessary.
Cost
$ 1,100 /线/会话MD安德森调查;$ 1,640 /线/会话MD安德森附属调查;$ 2,660 /线/会话用于其它外部调查
Time Line for Embryo Cryopreservation
Day 1
- 辖PMS以女性
- 辖HCG以女性和伴侣
- Transfer females to a separate cage
Day 6
- Flush oviducts to collect morula and freeze
- Thaw one straw for overnight culture
Day 7
- Count blastocysts in overnight culture
从GEMF获得服务
- 安排一个奥博金intment with Jan Parker-Thornburg by calling 713-795-2654, or e-mailjpthorn@mdanderson.org审查动物冷冻保存的类型和所需的动物数量
- Log in tohttps://mdanderson.ilabsolutions.com, register for an account, then go to the site to request service
- Read thePolicies and Proceduressection for important information regarding services rendered by the GEMF.
精子冷冻保存
We now utilize the EMMA protocol initially published by Naomi Nakagata as our method of sperm cryopreservation [Ostermeier GC等。2008.公共科学图书馆一3（7）：e2792。研究者将需要提供已根据由GEMF在收到在线申请的计划集以前交配两只雄性小鼠。每个男性的精子的十一秸秆将被冻结，并存储在GEMF。为了测试程序的完整性，我们将用一个吸管从每个男性与野生型卵母细胞，这将培养两个细胞阶段建立受精率进行IVF。
Cost
$ 1,100 /线/会话MD安德森调查;$ 1,640 /线/会话MD安德森附属调查;$ 2,660 /线/会话用于其它外部调查
时间线的精子冷冻保存
Day 1
- Two to four males are set up in mating to non-essential females supplied from the PI's colony
Day 5
- 男性未配合，使精子复苏
天10 11
- 男性被带到GEMF低温实验室的牺牲和精子复苏和冷冻保存
Approx. Day 17
- One sperm sample per male is thawed and mixed with unfertilized oocytes to allow forin vitrofertilization. After four hours, oocytes are washed and allowed to incubate overnight
Approx. Day 18
- Two-cell embryos resulting from the IVF are counted and the fertilization rate for the sperm sample is calculated. Project is complete
从GEMF获得服务
- Log in tohttps://mdanderson.ilabsolutions.com, register for an account, then go to the site to request service
- 甲GEMF技术人员将你与细节上的配对协议之前冷冻保存过程联系
- 你会在同一时间把正确的雄性交配的低温实验室事先安排与GEMF技师
- 该项目完成后，您会收到通知
IVF-冷冻保存
因为它往往是困难的调查产生的胚胎冷冻15-20肥沃的男性，我们还提供了IVF-冷冻保存程序的选项。对于此过程中，我们将需要两到四个优化育男性。男性将被处死，以获得精子，然后与卵母细胞混合in vitro. Two-cell embryos resulting will be vitrified and stored for future use. While this procedure is much easier on the investigator,那里经常会长时间等待这个服务时间，由于技术所需时间的长短. As in the embryo cryopreservation procedure, we will archive 200 embryos from a heterozygous x wild-type mating and 100 embryos from a homozygous x wild-type mating. All procedures will be tested by thawing a small aliquot of embryos and determining survival.
Cost
$1,700/line/session for MD Anderson investigators; $2,363/line/session for MD Anderson-affiliated investigators; $3,830/line/session for other outside investigators
时间线进行IVF-冷冻保存
Day 1
- Two to four males are set up in mating to non-essential females supplied from the PI's colony
Day 5
- 男性未配合，使精子复苏
天10 11
- Males are brought to the GEMF Cryo lab for sacrifice and sperm recovery, IVF and cryopreservation
Approx. Day 17
- 一个精子样品与未受精卵母细胞，以允许混合in vitrofertilization. After four hours, oocytes are washed and allowed to incubate overnight
Approx. Day 18
- Two-cell embryos resulting from the IVF are counted and the fertilization rate for the sperm sample is calculated. All two-cell embryos are vitrified and maintained in liquid nitrogen. One small group of embryos is thawed to determine survival rates. After successful vitrification of 200 embryos (100 embryos if a homozygous male is used), project is complete. (Note: If the requisite number of embryos is not obtained, the project will be repeated at an additional session-cost to the investigator.)
从GEMF获得服务
- Log in tohttps://mdanderson.ilabsolutions.com, register for an account, then go to the site to request service
- 一个GEMF技术人员将直到项目开始的等待时间的细节与您联系，并配合协议之前冻存程序
- 你会在同一时间把正确的雄性交配的低温实验室事先安排与GEMF技师
- 该项目完成后，您会收到通知