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Whole Mammary Gland Transplantation in Mice Protocol
小鼠全乳腺移植方案   

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Abstract

Whole Mammary Gland Transplantation involves transplanting an excised mammary gland into another, more suitable host. This method can be used to extend the life of a mammary gland past the mouse’s life span by transplanting the mammary gland of an older mouse into a young healthy mouse. As you can see in the video below (Video 1), by attaching it to the abdomen of the mouse, the gland will receive a steady blood supply and both epithelial and stromal cells will remain viable for up to one year. Although this method is not used often, it has been part of several experiments including determining whether the stroma or epithelium is the primary target in chemically induced mouse mammary tumorigenesis (Medina and Kittrell, 2005). To monitor transplants, palpate every week for tumor formation. The transplanted mammary gland may also be passaged serially every 8-10 weeks. Keep transplanted gland in the same mouse for no longer than one year.

Video 1. Whole mammary gland transplantation

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Keywords: Mammary gland(乳腺), Transplant(移植), Viable epithelial cells(存活上皮细胞), Mammary tumorigenesis(乳腺肿瘤发生)

Materials and Reagents

  1. Surgical gloves (Cardinal Health, catalog number: 2D72N80X )
  2. 1 ml Tuberculin (TB) syringe (BD, catalog number: 309625 )
  3. Dissolvable suture, size: 0000
  4. Animals: 8-12-week old female mice
    Note: Excised gland will need to be transplanted into the same strain of mouse. If donor and recipient are of different strains, the recipient must be immunocompromised (i.e., gland excised from Balb/c mouse and transplanted to NSG mouse).
  5. Nembutal sodium solution CII (Pentobarbital sodium injection, USP) (Akorn, Oak Pharmaceuticals, NDC 76478-501-20 )
  6. Ketofen® (Ketoprofen) (Zoetis, catalog number: 10004029 )
  7. 70% ethanol

Equipment

  1. Balance
  2. Hair clipper (Remington, model: PG525 )
  3. Hemostatic forceps (Roboz Surgical Instruments, catalog number: RS-7131L )
  4. Sharp/Ball Tip scissors (Fine Science Tool, catalog number: 14086-09 )
  5. Rat tooth forceps (Sklar Surgical Instruments, catalog number: 19-1260 )
  6. Curved forceps (Fine Science Tool, catalog number: 11052-10 )
  7. Heating pad (Sunbeam Products, catalog number: 000771-810-000U )
  8. Wound Clip applier, remover and clips (BD, catalog number: 427638 )

Procedure

  1. Mammary gland excision (not included in video)
    1. Using the same preparation for surgery and steps C1-C2 of the surgery procedure described below, locate the mammary gland to be excised and using curved forceps, make sure the whole gland is exposed and completely separated from the body cavity.
    2. Use rat tooth forceps to grasp the gland and using sharp scissors begin cutting the gland away from the skin until the whole gland is free from the skin and in one piece.
    3. Immediately transfer the excised gland to the recipient mouse and continue with step C3 of the surgery procedure.

  2. Prepare the mice for surgery
    1. Weigh mouse.
    2. Using 1 ml TB syringe, administer 40 mg/g body weight of Nembutal intraperitoneally to one side of the abdomen (this will provide 1 h of sedation).
    3. Wait until the mouse has fallen asleep; confirm by checking hind limb reflex by pinching foot.
    4. Use clippers to shave the abdomen.
    5. Using 1 ml TB syringe, administer 5 mg/kg body weight Ketofen® intraperitoneally on the opposing side of ketamine injection site. This is for pain relief.
    6. Place the mouse on a surgical board and secure limbs with surgical tape.
    7. Sterilize the abdomen with 70% alcohol.

  3. Surgery
    1. Using the ball tip scissors, make an inverted Y incision to expose the abdominal wall and inguinal fat pads. Begin on the midline slightly below inguinal nipple and extended up about 1 inch; extending the other two cuts between the fourth and fifth nipples on either side.
    2. Hold the skin with rat tooth forceps and using the curved forceps, separate the skin from the abdominal wall. Secure skin flaps with pins to expose abdominal wall.
    3. Place excised gland on the abdominal wall, on either side of the midline. Make sure gland is lying flat against the wall and is not twisted as seen below (Figure 1).


      Figure 1. Excised gland must lie flat against abdominal wall

    4. Using hemostatic forceps, curved forceps and suture, suture each end of the transplanted gland to abdominal wall as seen below (Figure 2).


      Figure 2. One suture in place at both ends of excised gland

    5. Reposition the skin flaps and close the incision with wound clips.

  4. Post-surgery
    1. Allow mouse to recover on heating pad set to lowest setting (~110 F). Allow time for the mouse to regain consciousness before returning to the cage (30 min-1 h).
      Note: Keep in mind that the sedation lasts 1 h.
    2. Administer 5 mg/kg body weight of Ketofen® intraperitoneally on either side of the abdomen 24 h later.
    3. Remove wound clips 7-10 days post-surgery.
    4. To monitor, palpate weekly for tumor formation.

Notes

Recipients were 8- to 12-week-old virgin female NSG, which were purchased from Jackson Laboratories. Animal experiments were conducted following protocols approved by the University of Kansas School of Medicine Animal Care and Use and Human Subjects Committee. All researchers must have this protocol approved with their own institution before preforming the procedure.

Acknowledgments

This protocol was developed in the Department of Molecular and Cellular Biology, Baylor College of Medicine and Department of Pathology and Laboratory Medicine, University of Kansas Medical School. The work was supported by NCI (1K99/R00 CA127462) to FB.

References

  1. Medina, D. and Kittrell, F. (2005). Stroma is not a major target in DMBA-mediated tumorigenesis of mouse mammary preneoplasia. J Cell Sci 118(Pt 1): 123-127.

简介

全乳腺移植涉及将切除的乳腺移植到另一个更合适的宿主中。该方法可用于通过将老鼠的乳腺移植到年轻的健康小鼠中来延长乳腺的寿命,超过老鼠的寿命。在下面的视频(视频1)中可以看到,通过将其附加到鼠标的腹部,腺体将获得稳定的血液供应,并且上皮细胞和基质细胞将保持活力长达一年。虽然这种方法不经常使用,但它已经是几个实验的一部分,包括确定基质或上皮是化学诱导的小鼠乳腺肿瘤发生的主要靶标(Medina和Kittrell,2005)。为了监测移植物,每周触诊肿瘤形成。移植的乳腺也可以每8-10周连续传播一次。将移植腺保持在同一只老鼠不超过一年。

关键字:乳腺, 移植, 存活上皮细胞, 乳腺肿瘤发生

材料和试剂

  1. 手术手套(Cardinal Health,目录号:2D72N80X)
  2. 1 ml结核菌素(TB)注射器(BD,目录号:309625)
  3. 可溶性缝合线,尺寸:0000
  4. 动物:8-12周龄雌性小鼠
    注意:切除的腺体需要移植到同一株鼠标中。如果供体和受体具有不同的菌株,受体必须免疫受损(即,从Balb / c小鼠切除并移植到NSG小鼠)。
  5. Nembutal钠溶液CII(Pentobarbital sodium injection,USP)(Akorn,Oak Pharmaceuticals,NDC 76478-501-20)
  6. Ketofen ®(酮洛芬)(Zoetis,目录号:10004029)
  7. 70%乙醇

    设备

    1. 平衡
    2. 吹风机(Remington,型号:PG525)
    3. 止血钳(Roboz Surgical Instruments,目录号:RS-7131L)
    4. 夏普/球尖剪刀(精细科学工具,目录号:14086-09)
    5. 大鼠牙镊子(Sklar Surgical Instruments,目录号:19-1260)
    6. 弯曲镊子(精细科学工具,目录号:11052-10)
    7. 加热垫(Sunbeam Products,目录号:000771-810-000U)
    8. 绕线夹施放器,拆卸器和夹子(BD,目录号:427638)

      程序

      1. 乳腺切除术(视频中不包括)
        1. 使用相同的手术准备和下述手术步骤C1-C2,定位要切除的乳腺并使用弯曲的钳子,确保整个腺体暴露并完全与体腔分离。
        2. 使用大鼠牙镊子抓住腺体,使用锋利的剪刀开始将腺体从皮肤上切开,直到整个腺体从皮肤上脱离出来,并一体成型。
        3. 立即将切除的腺体转移到受体鼠,并继续进行手术步骤C3
        4. 准备老鼠进行手术
          1. 称重鼠标。
          2. 使用1 ml TB注射器,腹膜内腹膜内给予40 mg / g体重的Nembutal(这将提供1小时的镇静)。
          3. 等到老鼠睡着了;通过踩脚检查后肢反射来确认。
          4. 用剪刀刮腹部。
          5. 使用1ml TB注射器,在氯胺酮注射部位的相对侧腹膜内施用5mg / kg体重Ketofen 。这是为了缓解疼痛。
          6. 将鼠标放在手术板上,用手术胶带固定四肢。
          7. 用70%酒精消毒腹部。

          8. 手术
            1. 使用球头剪刀,倒置Y切口,露出腹壁和腹股沟垫。从腹股沟乳头下方的中线开始延伸约1英寸;延伸两侧的第四和第五个乳头之间的另外两个切口。
            2. 用鼠齿镊子握住皮肤,并使用弯曲的镊子,将皮肤与腹壁分开。用针固定皮瓣以露出腹壁。
            3. 将切除的腺体置于腹壁两侧的腹壁上。确保腺体平躺在墙上,不会如下所示扭曲(图1)。


              图1.排泄的腺体必须平放在腹壁上

            4. 使用止血钳,弯曲镊子和缝合线,将移植腺的每一端缝合到腹壁,如下所示(图2)。


              图2.切除腺体两端的一条缝合线

            5. 重新定位皮瓣,并用伤口夹闭合切口。

            6. 手术后
              1. 允许鼠标在加热垫恢复到最低设置(〜110 F)。让老鼠重新恢复意识,然后返回笼子(30分钟到1小时)。
                注意:请注意,镇静剂持续1小时。
              2. 24小时腹腔内腹膜内给予5 mg / kg体重的Ketofen
              3. 手术后7-10天取出伤口夹。
              4. 监测,每周触诊肿瘤形成。

                笔记

                受试者为8至12周龄的处女女性NSG,购自Jackson Laboratories。堪萨斯大学医学院动物护理与使用与人体科学委员会批准的方案进行动物实验。所有研究人员必须在执行程序之前通过自己的机构批准该协议。

                致谢

                该协议是在堪萨斯大学医学院贝勒医学院和病理学和实验室医学系的分子和细胞生物学系开发的。这项工作得到NCI(1K99 / R00 CA127462)对FB的支持。

                参考

                1. Medina,D.和Kittrell,F.(2005)。 Stroma不是DMBA介导的小鼠乳腺发育不全的肿瘤发生的主要靶点。细胞科学118(Pt 1):123-127。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hansford, H., Hong, Y., Kittrell, F., Medina, D. and Behbod, F. (2017). Whole Mammary Gland Transplantation in Mice Protocol. Bio-protocol 7(11): e2326. DOI: 10.21769/BioProtoc.2326.
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