小鼠的存活在宫内手术,可在开发过程中基因表达的分子操纵。在这里,我们描述了使用高频率的超声波成像引导逆转录病毒载体注射到鼠脑在胚胎一天(五)9.5。
小鼠的存活在宫内手术,可在开发过程中基因表达的分子操纵。然而,由于子宫壁是不透明的,在早期胚胎发育,极大地限制的能力为目标的特定部位的显微注射胚胎。幸运的是,高频率的超声波成像许可证生成可用于实时图像引导胚胎地区利益的一个显微注射针。在这里,我们描述了利用这种成像引导胚胎一天鼠标的前脑的脑室系统(E)9.5逆转录病毒载体注射。这个方法使用一个允许访问的子宫角,和一个特别设计的板,允许主机的胚胎,而他们的成像和注射生理盐水中沐浴的剖腹探查。成功的手术,往往会造成大部分或全部注入的胚胎存活到任何利益随后的时间点(embryonically或产后)。这里所描述的原则,可以稍作修改用于执行注射到E8.5胚胎羊水(从而允许尚未闭合的神经管,后沿前程度的感染),或进入脑室系统大脑在E10.5/11.5。此外,在中期源性年龄(〜E13.5),超声显像可用于直接注射到病毒感染或细胞移植特定的大脑区域。使用超声显像在小鼠子宫内注射,以指导是一个非常强大的技术许可证,否则将异常艰难,如果不是不可能的方式操纵分子和细胞的小鼠胚胎。
使用超声引导下的病毒注射液可进行E8.5 – E11.5,其中只有羊膜囊,可以在E8.5注射,而两个羊膜囊和脑室系统,可以从E9.5 – E11.5注入。正确执行时,病毒颗粒注入脑室系统使用超声引导下进入脑室系统的上皮细胞,因此,可以感染前脑(大脑皮层,基底节,间脑,眼等),中脑的各种结构和后脑(Figure1C)。注入羊膜囊病毒进入细胞胚胎的外部。除了病毒,还可以用于超声引导下注入到发育中的胚胎细胞。因此,这种技术提供了多种选择,从设计实验时,选择(一段时间的发展,结构,病毒/细胞)。
至关重要的是,注射的病毒或细胞表达病毒/注射细胞易于识别的记者。从历史上看,人类胎盘碱性磷酸酶(PLAP)或GFP已被使用。从以往的经验,我们发现PLAP可以是一个非常有用的报告基因,因为PLAP组织化学和免疫组织化学染色产生一个尖锐而鲜明的单细胞染色,让来分析细胞的形态和位置,并进行克隆分析。由于病毒GFP的表达可以是淡淡的,GFP的记者可能不理想的染色组织,但有用的,如果想获得单个细胞,通过荧光激活细胞分选(FACS)。
The authors have nothing to disclose.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Micromanipulator | Narishige | 91084361683D | ||
HI-7 pipette holder | Narishige | 9108436141 | ||
10 mm support arm | Carl Zeiss | 9108436206 | ||
IP iron base plate | Carl Zeiss | 9108436167 | ||
CI-1 connector for pipette holder | Carl Zeiss | 9108436257 | ||
1mm ID tubing | Carl Zeiss | 911013 | ||
3-way luer lock stopcock (pkg 10) | Carl Zeiss | K420163-4503 | ||
Motor controller, 2-4 axes | Newport | 860-c2 | ||
Joystick | Newport | PMC200-J | ||
Micrometer adapter | Newport | ADAPT-BM17-375 | ||
Motorized drive | Newport | CMA-25CC | ||
Cable assembly | Newport | 860I-10 | ||
25 mm translation stage | Newport | UMR8-25 | ||
Micrometer | Newport | BM17-25 | ||
Base plate | Newport | M-PBN8 | ||
Inner mounting bracket | Newport | EQ80-I | ||
Outer mounting bracket | Newport | EQ80-E | ||
Man. infusion/withdrawl pump | Stoelting | 51222 | ||
25 μl microsyringe | Stoelting | 51113 | ||
24″x12″x0.5″ metric board plate | Edmund | NT03-640 | ||
Set of self-adjusting feet | Edmund | NT34-841 | ||
Bar-type lens holder | Edmund | NT03-669 | ||
Right angle post clamp | Edmund | NT53-357 | ||
2″ post holder | Edmund | NT03-646 | ||
4” steel post | Edmund | NT36-499 | ||
6” steel post | Edmund | NT36-503 | ||
12/pk set screws, 1/4-20 x 1/2″ | Edmund | NT03-644 | ||
Additional Materials: | ||||
PBS, pH7.4 | Invitrogen | 10010049 | ||
Slide warmer | Fisher Scientific | Numerous options | ||
Double Edge Razor Blades | Electron Microscopy Sciences | 72000 | ||
Borosilicate Glass O.D: 1.0mm, I.D.: 0.05mm | Sutter Instruments | B100-50-10 | ||
1cc Insulin Syringe U-100 27G5/8 | Becton Dickinson | 329412 | ||
Autoclip Applier | Becton Dickinson | 427630 | ||
Autoclips, 9mm (to close skin incision) | Becton Dickinson | 427631 | ||
5.0 Silk sutures | Fisher | S-1173 | ||
Sterilube ophthalmic ointment | Fera pharmaceuticals | 48102-012-35 | ||
MB-10 or equivalent disinfectant | Quip labs | |||
10% bleach | Multiple sources |
“One of the following ultrasound backscatter microscopes can be used for this procedure. Although in the video the P40 from Paradigm-Medical is used, the P40 is no longer commercially available, and has been replaced by the P60, which is similarly priced and can be used for the same application. The additional materials described for stage construction are for use with Paradigm-Medical device(s). The Vevo 2100 comes with equipment for small animal imaging.
Anesthetic and Analgesic
The Nembutal solution (50 mg/mL injectable sodium pentobarbital; produced by Abbott laboratories) can be obtained from Bulter Schein Animal Health. Note that this is a schedule II controlled substance and requires a Drug Enforcement Agency (DEA) license to obtain.
The Bupivacaine solution (0.25%; produced by Hospira, Inc, among others) can be obtained from a number of distributors, including Moore Medical.