Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

Summary

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

Abstract

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Introduction

Embryo transfer is probably the most frequent surgical procedure performed in the mouse model. This technique is essential to obtain offspring from embryos subjected to in vitro manipulation techniques and, therefore, constitutes a necessary step for the development of genetically modified models by pronuclear injection, lentiviral transduction, or chimera formation. Besides, the technique allows the study of the developmental effects of diverse insults occurring during preimplantation development. The use of artificial reproduction techniques¹ or the exposure to abnormal concentrations of different substances or metabolites² may affect ....

Protocol

All animal experiments were approved by the Beltsville Area Animal Care and Use Comittees (BAACUC 11-015) in accordance with USDA Animal Care and Use Guidelines.

1. Anesthesia and Analgesia (Common for Both Surgical Procedures)

1. Weigh the mouse and load the following anesthetics and analgesic in two 1 ml syringes with 27 G needles:
   1. Ketamine (0.1 mg/g: 0.01 ml/g of a 10 mg/ml solution) and xylazine (0.01 mg/g: 0.005 ml/g of a 2 mg/ml solution).
   2. Buprenorphine (0.1 μg/g: 0.01 ml of a 0.01 mg/ml solution).
2. Immobilize the mouse by picking up the scru....

Results

Utero-tubal embryo transfer provides a mean to transfer embryos to the uterus avoiding some of the complications associated to the uterine embryo transfer^2,9,10. In Table 1 we show some representative result we have obtained transferring CD1 blastocysts subjected to different kinds of manipulations to CD1 recipients following the protocol described. The survival to term (% of embryos resulting in a pup) or survival to E15 (in the case of lentivirus exposed) is similar between embryos simply cu.......

Discussion

Vasectomy is a relatively straight forward surgical technique that does not involve major difficulties. When sanitizing with povidone iodine and ethanol make sure that the last wash (with ethanol) removes povidone iodine, as it may irritate the peritoneum. The access to vas deferens can also be achieved by the scrotum or performing a transversal incision in the abdomen⁸. Scrotal incision has been recommended to transversal abdominal incision due to the comparatively smaller incision needed and slightl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine	VEDCO	Ketaved ANADA 200-257	To be ordered by a licensed veterinarian.
Xylazine	Lloyd Laboratories	Anased NADA #139-236	To be ordered by a licensed veterinarian.
Buprenorphine	Generic	NDC 400-42-010-01	To be ordered by a licensed veterinarian.
Eye ointment	Novartis	Genteal
Antibiotic	Pfizer	Clavamox NADA #55-101.	Added to drinking water (0.3 mg/ml of amoxicillin trihydrate and 0.075 mg/ml of clavulanate potassium). Add 1.5 ml of the reconstituted 15 ml bottle to 250 ml of water.
Dressing serrated forceps	ROBOZ	RS-8120	Any medium size surgical-grade steel straight forceps will work.
Microdissecting serrated forceps	ROBOZ	RS-5137	These ones are curved at a 90º angle. Straight forceps can be used if preferred.
Slight curved micro dissection forceps	ROBOZ	RS-5136	This model is particularly useful to hold the oviduct.
Scissors	ROBOZ	RS-5880	Any regular surgical grade steel small straight scissors will work.
27 G needles	Beckton-Dickinson	305136	Smaller needles (30 G) can be also used. 25 G may be a bit too big.
Clip applier	MiKRon	42763
9 mm Clips	MiKRon	427631
Clip remover	MiKRon	7637	Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead.
Suture needle holder	ROBOZ	RS-7820
Suture	Dowist Gell	5-0 Dexon S 7204-21	Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle.
Glass capillaries	VWR	100 ul calibrated pipettes 53432-921	It includes a mouth aspirator system that only requires to attach a 0.22 µm filter in the tubing to be ready to use. More information can be obtained in Nagy et al.8
Burner	KISAG AG	Typ 2002	Gas operated burner, can be charged with Kigas (CH-4512, from the same vendor). Alcohol burners may be also used, but gas provides a higher temperature and this burner provides a small and precise flame.
Stereomicroscope	Leica	MZFLIII	This is an expensive estereomicroscope with fluorescence, that can be also used for other purposes. There are cheaper options such as Leica MZ8 or Nikon SMZ-10 or SMZ-2B, to name a few. It is better to use two stereomicroscopes, one for handling the embryos (which does not need to be a very nice one) and another one for the recipient. The one used for the recipient should display a long distance from the stage plate to the objective lense, in order to be able to focus 3-4 cm above the stage plate (where the oviduct will be placed) and still leave some room for the surgeon; most of the stereomicroscopes can do this, but some cannot.
Fiber optics ilumination	Dolan Jenner	Fiber lite	To iluminate the surgical area. There are different systems available.
Warm stages	American scope	http://store.amscope.com/tcs-100.html	These can be placed over the stage plate of the stereomicroscope. Some modifications (inserting a stick to level the stage) may be needed if it is too short for the stereomicroscope. A big warm stage can be used for warming the cage if it is available. If not, a regular heating pad can be used, but temperature must be checked.
Culture dishes for embryo manipulation	Falcon	353001	351008 may be also used; they made narrower drops.