Efficient method for transfer of microinjected eggs to mouse ampulla for generating transgenic mice

Wen et al. SpringerPlus (2016) 5:2076 DOI 10.1186/s40064-016-3760-6 Open Access METHODOLOGY Efficient method for transfer of microinjected eggs to mouse ampulla for generating transgenic mice Guang Wen1*, Jing Di2, Qian Li3, Jianling Chen3, Ling Jin3, Cheng Wang3 and Sanqing Xu1,4 Abstract Background: The new method described here is highly efficient in transferring microinjected mouse eggs (MEs) through the bursa membrane of a surrogate mother mouse to the ampulla of the oviduct without damaging the blood vessels on the bursa membrane. Results: This method causes no loss of blood, and it produces newborn pups/founders from approximately 70% of the transferred MEs, because only a small hole is made on the blood vessel–free area of the bursa membrane and ampulla of the surrogate mother mouse. The infundibulum remains intact. The small hole on the bursa membrane/ ampulla may already heal up before the delivery of the new pups. The method described here consists of a simple operation with a home-assembled drill head holding a self-closing fine forceps on one end, while the drill head assembly body is hooked up with the light housing clamp of a dissecting light microscope. The drill head assembly body can be alternatively hooked/tied up to an appropriate size of clamp (purchased from Home Depot) screwed to any light stand with folding segments. Conclusion: This system is able to steadily hold the self-closing fine forceps without shaking and to let the operator use their two hands to steadily hold and quickly insert the pipet carrying the MEs into the ampulla without any delay. Generally MEs stay alive for approximately 15 min at room temperature. The shorter the insertion time is, the more MEs that will survive. Thus, this method may produces more pups/founders. Keywords: New device, Microinjection of eggs, Transgenic/knockout mice, Generation of transgenic/knockdown mice Background In the current method for producing transgenic/knockdown mice, the bursa membrane of a surrogate mother mouse is generally opened or torn apart to expose the infundibulum before a pipet tip containing microinjected mouse eggs (MEs; eggs also referred to as embryos) is transferred/inserted through the top opening of the infundibulum to the ampulla of the surrogate mouse (Behringer et al. 2014; Wen and Chen 2004; Hogan et al. 1994). This task is rather challenging, because blood *Correspondence: ; 1 Department of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Full list of author information is available at the end of the article from the blood vessels of the bursa membrane floods and covers up the infundibulum opening and ampulla. As a result, it eventually becomes impossible for the operator to see the top opening of the infundibulum/ ampulla before inserting the pipet tip to transfer the MEs (Behringer et al. 2014; Wen and Chen 2004). Because the bursa membrane and oviduct are slippery, it is difficult to punch a small hole through them with a 29-gauge needle or any small needle without holding the bursa membrane and infundibulum together with a fine, self-closing forceps (Liu et al. 2013), because the bursa membrane and ampulla will move around. The new method described here overcomes these two problems. In addition, because our device holds the bursa membrane and infundibulum stably with self-closing forceps, it frees the investigator’s © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wen et al. SpringerPlus (2016) 5:2076 two hands to perform a quick transfer/injection of the MEs to the ampulla. The shorter the transfer/time, the higher the survival rate of the MEs at room temperature. It is therefore easy to achieve newborn pups/founders from 70% of these MEs. Methods Animals Mice of CD-1 strain (Mus musculus) were purchased from the Charles River Laboratory (Wilmington, MA). Mice were cared for and maintained humanely in our animal colony, under proper housing and husbandry conditions conforming to the U.S. Public Health Service guidelines for humane care and use of laboratory animals of the National Research Council (2011) and the National Institutes of Health (1985). All of the new and used cages, air filters, water feeding bottles, and other items used to care for these animals were cleaned and autoclaved, as a pathogen-free environment is required for every animal in our animal colony. In addition, this study was part of our project entitled “Production and Breeding of Neuroligin-3 and -4 Knock Down Mice for Autism Research,” which was approved by the Animal Welfare Committee of our Institute (PR#330). The mice were serologically tested by Charles River Laboratory to confirm the absence of the following pathogens: Sendai virus, pneumonia virus of mice, mouse hepatitis virus, minute virus of mice, Theiler’s virus, reovirus, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, ectromelia virus, mouse pneumonitis virus, polyoma virus, mouse adenovirus FL/K87 1 & 2, epizootic diarrhea of infant mice virus, mouse cytomegalovirus, Hantaan virus, Encephaokitozoon cuniculi, ciliaassociated respiratory bacillus, mouse parvovirus, and mouse thymic virus. Mice were housed in a quarantine facility at our animal colony with a 12-h light–12-h dark cycle and 10–15 air changes/h. Mice were housed on sterilized corncob bedding (Harlan, Indianapolis, IN) in sterilized cages (polysaufone Standard mouse cage, ACE, Allentown, PA) equipped with stainless steel wire bar tops and filtered cage tops. Mice were fed with Lab Diet 5015, Mouse Diet (PMI Nutrition International, Inc., Brentwood, MO) and hyperchlorinated water available ad libitum in bottles. Cage litter was changed every seven days in a class-II biological safety cabinet in the animal colony. Superovulation in female mice around 28 days old was achieved by intraperitoneal (i.p.) administration of 5–7.5 IU (=0.1–0.15 ml of 50 IU/ml) of pregnant mare’s serum/ml (Sigma-Aldrich, St. Louis, MO) 47 h prior to i.p. administration of 5–7.5 IU (=0.1–0.15 ml of 50 IU/ml) of human chorionic gonadotropin/ml (Sigma-Aldrich, St. Louis, MO). Each female mouse was Page 2 of 5 then placed in a cage with a fertile stud male mouse at around 4–5 p.m.; the male and female mice were left together overnight. The presence of a copulation plug in the vagina the next morning generally indicated that the female mouse was carrying fertilized eggs/oocytes. All mice were sacrificed by i.p. administration of an overdose (>0.017 ml/body weight in grams) of 2.5% Avertin anesthetic (Sigma-Aldrich). Fertilized eggs (i.e., (...truncated)