to BioTechniques free email alert service to receive content updates.
A device for the simple and rapid transcervical transfer of mouse embryos eliminates the need for surgery and potential post-operative complications
 
1The Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA
2ParaTechs Corp., University of Kentucky, Lexington, KY, USA
3Department of Microbiology, Immunology, & Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA
BioTechniques, Vol. 47, No. 5, November 2009, pp. 919–924
Full Text (PDF)
Supplementary Material
Abstract

We describe a novel device that can be used for the transcervical transfer of embryos into pseudopregnant female mice. This nonsurgical embryo transfer (NSET) device is as efficient as standard surgical embryo transfer in the production of transgenic mice, and can also be used for the transfer of embryonic stem cell–containing chimeric blastocysts and cryopreserved embryos. In addition to the elimination of surgery, recipient females do not have to be anesthetized. The NSET device eliminates a painful surgical procedure as well as potential complications associated with anesthesia/post-operative care, reduces the technical expertise and equipment needed for surgical transfer, and represents substantial cost savings and regulatory reduction. NSET technology provides an easy and rapid alternative to surgical embryo transfer.

Introduction

The ability to manipulate the mouse germline via gene transfer and targeted gene modification has dramatically advanced the use of mice in biomedical research (1,2,3). However, these strategies of genetic manipulation require the surgical transfer of embryos and lead to pain and distress for the females that serve as embryo recipients. In addition, potential problems with anesthesia and post-operative recovery, and possible post-surgical infection, require that considerable time be spent to monitor these recipient females (4,5). Furthermore, specialized surgical equipment, the need for some facilities to perform sterile procedures, and the substantial training needed for proficiency in uterine and oviduct surgery restrict the number of institutions where such procedures can be performed (6). The time and effort spent with paperwork required for compliance with regulatory guidelines is also an issue with surgical embryo transfer in rodents. To eliminate many of these problems, we have developed a device that can be used for the transcervical transfer of embryos into pseudopregnant female mice, which we call a nonsurgical embryo transfer (NSET) device.

Using a handmade device, our data indicate that NSET-mediated embryo transfer is as effective as surgical embryo transfer. Based on prototype devices, we have manufactured an NSET device that is sterile, more uniform in size, and more stable than the handmade device. NSET technology results in a dramatic reduction in the time and expertise required for the production of genetically modified mice. In addition, this technology eliminates surgery and the potential complications associated with anesthesia/post-operative care, and represents substantial cost savings and regulatory reduction.

Materials and methods

Mice

C57BL/6 X C3H F1/Hsd (B6C3F1), C57BL/6N/Hsd (BL/6), and Crl:CD1(ICR) (CD-1) mice were purchased from Charles River (Frederick, MD, USA) or Harlan Laboratories (Indianapolis, IN, USA). Mice were housed in positive individual ventilation (PIV) or microisolator cages with water and chow provided ad libitum and maintained on a 14 h/10 h light/dark cycle. All experimental procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee, following guidelines established by the National Institutes of Health.

Embryos and embryonic stem (ES) cells

Embryos were removed from pregnant female mice that had been superovulated using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and mated to males using standard conditions as described (6). Embryos were cultured in potassium simplex optimized medium (KSOM) media (Cat. no. MR-106-D; Millipore, Billerica, MA, USA). The R1 ES cell line was obtained from Dr. Andras Nagy (Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada), cultured on mouse embryo fibroblasts and used for the production of chimeric embryos by embryo aggregation (7).

NSET devices

Handmade prototype NSET devices were generated using P-2 pipet tips (Model no. 4826; Corning, Corning, NY, USA) and BD Angiocath I.V. catheters (Becton-Dickinson, Sandy, UT, USA). The P-2 pipet tip was inserted into the catheter (Supplementary Figure 1A and 1B) or the catheter tip was glued to the end of the P-2 pipet tip (Supplementary Figure 1C). The polyolefin speculum is 0.4 in (1.016 cm) in length. Initial testing was performed on euthanized female mice. Based on these results, further studies were performed on live animals; the data described in this paper were generated using handmade NSET devices. The manufactured NSET device was developed by ParaTechs Corp. (Lexington, KY, USA) based on results with prototype devices. Using specifications provided by ParaTechs Corp., the devices were produced by Martech Medical Products (Harleysville, PA, USA). The catheter tip is extruded from Teflon-FEP (DuPont, Wilmington, DE, USA) and the hub component is molded with polyethylene (a prototype is shown in Figure 1). The length of the tip and hub are 1.025 in (2.60 cm) and 0.625 in (1.588 cm), respectively. The outside and inside diameters of the tip are 0.03 in (0.076 cm) and 0.02 in (0.051 cm), respectively, and the tip is tapered at the end. The speculum is extruded from polyethylene and is 0.4 in (1.016 cm) long with outside and inside diameters of 0.162 in (0.411 cm) and 0.140 in (0.356 cm), respectively. The single-use NSET device and speculum are packaged in a clean room in a Tyvek (DuPont) pouch, followed by ethylene oxide sterilization, and are commercially available from ParaTechs Corp. (Cat. no. 859–218–6541). Details about embryo transfer using NSET technology can be found in the Supplementary Materials.

  1    2    3