Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery
使用慢病毒递送的离体滋养层特定基因操作   

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Proceedings of the National Academy of Sciences of the United States of America
Nov 2016

 

Abstract

In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of ‘gain-of-function’ and ‘loss-of-function’ blastocyst manipulations can be evaluated with in vitro outgrowth assays or following transfer to pseudopregnant rats.

Keywords: Blastocyst (囊胚), Placenta (胎盘), Trophoblast (滋养层), Lentiviral vector (慢病毒载体)

Background

The placenta functions as a conduit between the mother and the developing fetus and is essential for viviparity (Georgiades et al., 2002). It is specialized to facilitate and coordinate maternal adaptations to pregnancy and fetal development (Soares et al., 2014; Burton et al., 2016). The placenta contains trophoblast cells, which perform several specialized functions. Acquisition of trophoblast cell specializations requires highly regulated differentiation of trophoblast stem and progenitor cell populations (Maltepe and Fisher, 2015). The mature rat placenta is comprised of two morphologically and functionally distinct compartments: the junctional zone and the labyrinth zone (Soares et al., 2012). Progenitor cells, spongiotrophoblast cells, glycogen trophoblast cells, and polyploid trophoblast giant cells comprise the junctional zone. An invasive trophoblast lineage arises from progenitors within the junctional zone region (Ain et al., 2003; Soares et al., 2014). During the last week of gestation, these cells move out of the placenta and invade into the maternal uterine mesometrial compartment (Ain et al., 2003; Pijnenborg and Vercruysse, 2010). The innermost layer of the placenta (proximal to the developing fetus) is called the labyrinth zone, which consists of trophoblast cells and fetal vasculature derived from the allantois. Progenitor trophoblast cells within the labyrinth zone fuse to form syncytia, which provide barriers between maternal and fetal compartments (Soares et al., 2012). The labyrinth zone consists of an elaborate branched structure, providing a large surface area for nutrient, waste and gas exchange with the fetus (Knipp et al., 1999; Watson and Cross, 2005).

Specific modifications of the trophoblast lineage can be achieved using lentiviral transduction of the outer layer of the embryo at the blastocyst stage, termed trophectoderm (Georgiades et al., 2007; Malashicheva et al., 2007; Okada et al., 2007; Lee et al., 2009) (Figure 1). This method allows for efficient manipulation of all trophoblast cell lineages. It also facilitates discrimination between trophoblast and embryonic contributions to phenotypic outcomes arising from global genetic manipulations. The experimental basis for selective trophoblast viral infection is directly related to the structure of the blastocyst and the tight epithelium formed by the outer trophectoderm, which effectively restricts viral particle access to the inner cell mass (Georgiades et al., 2007; Malashicheva et al., 2007; Okada et al., 2007; Lee et al., 2009). This method can be utilized to investigate the consequences of both ‘gain-of-function’ and ‘loss-of-function’ manipulations of the trophoblast lineage, which can provide insights into molecular mechanisms controlling placental development (Georgiades et al., 2007; Malashicheva et al., 2007; Okada et al., 2007; Lee et al., 2009; Morioka et al., 2009; Kent et al., 2011; Chakraborty et al., 2016; Muto et al., 2016).


Figure 1. Schematic showing experimental plan for lentiviral transduction of blastocysts and downstream analyses. Blastocysts are transduced with lentiviral particles (shown in green) expressing specific ‘gain-of-function’ or ‘loss-of-function’ constructs and cultured for 72 h for outgrowth assays or directly transferred to day 3.5 pseudopregnant animals for in vivo analyses.

Materials and Reagents

  1. Multi-well glass agglutination plates (Scientific Device Laboratory, catalog number: 074-3032B )
  2. Tissue culture plate, 6-well (MIDSCI, catalog number: TP92006 )
  3. Needle,18 gauge (BD, BD Biosciences, catalog number: 305195 )
  4. Syringe, 1 ml
  5. Tissue culture plate, 12-well (MIDSCI, catalog number: TP92012 )
  6. Glass mouth pipette (Drummond Scientific, catalog number: 2-000-100 )
  7. Aspirator tube assembly, 15” (Drummond Scientific, catalog number: 2-000-000 )
  8. Tissue culture plate, 4-well (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 176740 )
  9. Plastic non-tissue culture Petri plates (Fisher Scientific, catalog number: AS4052 )
  10. Female rats (8-10 weeks of age)
  11. Lentiviral packaging reagents: pRSV-Rev (Addgene, catalog number: 12253 ), pMDLg/pRRE (Addgene, catalog number: 12251 ), VSVG envelope plasmid (pMD2.G, Addgene, catalog number: 12259 )
  12. Sterile saline solution (0.9% Sodium Chloride)
  13. Opti-MEM culture medium (Thermo Fisher Scientific, catalog number: 51985034 )
  14. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668-027 )
  15. p24 enzyme-linked immunoassay kit (Takara Bio, Clontech, catalog number: 632200 )
  16. M2 medium (Millipore Sigma, catalog number: MR-015-D )
  17. KSOM medium (Millipore Sigma, catalog number: MR-121-D )
  18. Acid Tyrode’s solution (Sigma-Aldrich, catalog number: T1788 )
  19. Mineral oil (Sigma-Aldrich, catalog number: M8410 )
  20. 4% paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  21. RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  22. Fetal bovine serum, heat inactivated (Sigma-Aldrich, catalog number: F2442 )
  23. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  24. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  25. Penicillin, and streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  26. (Optional) Clontech LentiX titration kit (Takara Bio, Clontech, catalog number: 631235 )
  27. Outgrowth culture medium (see Recipes)

Equipment

  1. Centrifuge (Beckman Coulter, model: OptimaTM L-100XP , catalog number: 392050)
  2. Standard inverted light microscope or stereo microscope
  3. Standard CO2 cell culture incubator, 5% CO2, 37 °C

Software

  1. ImageJ software (NIH)

Procedure

  1. Preparation of rats for timed pregnancy
    1. Animals are maintained in an environmentally-controlled facility with lights on from 06:00 to 20:00 (14 h light:10 h dark cycle) and are allowed free access to food and water.
    2. Female rats (8-10 weeks of age) are placed with male rats (> 3 months of age) of the same strain.
    3. Confirmation of mating is determined by inspection of vaginal lavages. Plastic pipettes are loaded with sterile saline solution. Saline is delivered to the vagina. Vaginal lavages are transferred to clean multi-well glass plates and observed under a microscope. The presence of sperm in the vaginal lavage is considered gestation day (gd) 0.5.
    Notes:
    1. In the rat, vaginal retention of seminal plugs is not common. Thus, it is necessary to obtain vaginal lavages as opposed to visual identification of seminal plugs.
    2. Lavages should be obtained between 08:00-10:00 for confirmation of sperm. If collected later, sperm may not be easily visible in the vaginal lavage and may lead to a false negative.
    3. Plastic pipettes should be washed thoroughly before obtaining lavages, so that there is no sperm contamination from one animal to the next, which may lead to a false positive.

  2. Virus production and concentration
    1. 293FT cells are split into 6-well plates at 70-80% confluency.
    2. After 16-20 h post plating, the medium is changed to Opti-MEM medium at 1.5 ml per well.
    3. Proceed to transfection 1 h following the medium change.
    4. Lentivirus is produced following transient transfection of a transducing vector and third generation packaging system plasmids. Prepare transfection mix utilizing Lipofectamine 2000 following manufacturer’s protocol. Add 500 ng of the transducing vector (shRNA construct or overexpression construct), 200 ng of pRSV-Rev (Addgene plasmid 12253), 500 ng of pMDLg/pRRE (Addgene plasmid 12251) and 300 ng of VSVG envelope plasmid (pMD2.G, Addgene plasmid 12259).
    5. Transfection mix is removed after 8 h, and 2 ml of Opti-MEM medium supplemented with 5% fetal bovine serum is added.
    6. The medium is removed after 20 h and collected in tubes stored at 4 °C. Two collections are performed in a period of 40-48 h.
    7. Centrifuge the culture supernatant to remove cell debris, filter sterilize and concentrate by ultracentrifugation (35,000 x g for 3 h). Store at -80 °C until used for transduction. Do not freeze-thaw.
    8. Lentiviral vector titers should be determined by measurement of p24 gag antigen by enzyme-linked immunoassay.

  3. Embryo collection
    1. Reproductive tracts of gd 4.5 pregnant rats are collected between 08:30 to 09:30.
    2. Uteri are dissected from adipose tissue and removed by cutting at the utero-tubal junction and cervix.
    3. Uteri are flushed with M2 medium from the cervical end with an 18-gauge needle connected to a 1 ml syringe.
    4. The flushed exudate is collected in a well of a 12-well tissue culture plate.
    5. Blastocysts are retrieved through a mouth pipette and transferred to a droplet of KSOM medium.
    6. Blastocysts are maintained at 37 °C in a CO2 incubator for 30 min to stabilize from the handling-associated stress.
    Notes:
    1. The timing of collection is very important for obtaining blastocyst stage embryos. If collected too early, the embryos will be in the oviduct and no blastocyst stage embryos will be obtained by flushing the uterus. If the collection is done too late, the embryos begin to attach to the uterus and it becomes very difficult to retrieve them by flushing the uterus.
    2. Embryos should be immediately transferred to KSOM medium at 37 °C in a 5% CO2 incubator and not left in unbuffered medium for extended periods.
    3. For details regarding embryo flushing and collection, please refer to Chiu et al., 2010.

  4. Removal of zona pellucida from blastocysts
    1. Place two separate droplets of Acid Tyrode’s solution (approximately 50 µl droplet size) in a Petri dish.
    2. In a second Petri dish, prepare 5 individual droplets of KSOM medium (approximately 40 µl droplet size).
    3. Under an inverted microscope, blastocysts are transferred to the first Acid Tyrode’s solution droplet and zona pellucida integrity monitored. If the zona pellucida dissolution is incomplete, then pipette the blastocysts into the second droplet and then immediately transfer them to the KSOM medium droplets.
    4. After successful zona pellucida removal and 5 KSOM washes, transfer the blastocysts into KSOM droplet covered with mineral oil. Mineral oil should completely cover the KSOM droplet and the total volume of mineral oil necessary will depend on the Petri dish size (2-3 ml will be sufficient to cover the KSOM droplet).
    5. Incubate the blastocysts at 37 °C for further stabilization.
    Notes:
    1. The survival of the embryo depends on the incubation time and exposure to Acid Tyrode’s solution. Overexposure to Acid Tyrode’s solution leads to disrupted and collapsed embryos, which will not be suitable for outgrowth or in vivo transfer experiments.
    2. When embryos are transferred to Acid Tyrode’s solution, constant observation is necessary to remove them immediately when the zona pellucida begins to dissociate. Once the zona pellucida is removed, the embryos should be immediately washed and transferred to a KSOM droplet.
    3. After removal of zona pellucida, embryos become sticky and tend to aggregate. Careful and gentle mouth pipetting is recommended to detach the embryos and proceed with further wash steps.

  5. Virus incubation and washes
    1. Lentiviral particles containing ‘gain-of-function’ or ‘loss-of-function’ constructs are generated, concentrated and stored, as previously reported (Lee et al., 2009).
    2. Add 10 µl of concentrated virus in a 30 µl KSOM droplet.
    3. Transfer blastocysts into the KSOM droplet containing virus particles. The droplet should be covered with mineral oil (2-3 ml will be sufficient to cover the KSOM droplet).
    4. Incubate the blastocysts in virus at 37 °C for 4 h.
    5. After the incubation, wash the blastocysts sequentially in 10-KSOM droplets.
    6. After the 10th wash, transfer the blastocysts into a droplet of KSOM covered with mineral oil and incubate at 37 °C for 15 min.
    Notes:
    1. The p24 coat protein measurement gives a relative assessment of virus concentration and does not necessarily indicate efficacy of infectivity of the virus batch.
    2. Alternative qPCR approaches (Clontech LentiX titration kit) can also be utilized to measure virus titers. 1 x 108 through 3 x 108 infectious unit/ml can be used to incubate blastocysts for effective transduction.
    3. Virus concentrations of 500 ng of p24/ml have been effective for transduction. However, effective viral particle concentrations need to be empirically determined for each experimental application.

  6. Preparation for blastocyst outgrowth
    1. In a single well of a 4-well tissue culture plate, add 750 µl of outgrowth culture medium (see Recipes).
    2. Add a single virally-manipulated blastocyst to each well and incubate at 37 °C.
    3. Observe the blastocysts daily, until the blastocysts have attached to the 4-well tissue culture plate.
    4. Once the blastocysts are attached, remove the medium and add fresh medium. Usually, it takes 2-3 days for the blastocysts to hatch and attach.
    5. On day 5 of incubation, outgrowth will be observed in control blastocysts (Figure 2).
    6. Outgrowths can be fixed in 4% paraformaldehyde for immunofluorescence, or can be harvested for further biochemical/molecular analysis.


      Figure 2. Effects of oxygen tension and KDM3A expression on blastocyst outgrowth. A. Schematic showing experimental plan for lentiviral transduction of blastocysts and outgrowth assay. Blastocysts were transduced with control (Ctrl) or Kdm3a shRNA and cultured for 72 h to allow hatching from the zona pellucida. The attached blastocysts were exposed to ambient (Amb) or low oxygen (0.5% O2) for 24 h and analyzed. B. Representative images of blastocyst outgrowths from Ctrl shRNA and exposed to Amb, Ctrl shRNA and exposed to 0.5% O2, and Kdm3a shRNAs and exposed to 0.5% O2. C. Measurement of Kdm3a transcripts in control and knockdown cultures was measured by qRT-PCR. Asterisks indicate significant differences among groups (n = 6/group; *P < 0.05). D. The bar graph shows quantification of outgrowth area in square millimeters. The area of the outgrowth was measured using ImageJ software (Ctrl shRNA + Amb, n = 6; Ctrl shRNA + 0.5% O2, Kdm3a shRNA1 + 0.5% O2, n = 10; Kdm3a shRNA2 + 0.5% O2, n = 10; *P < 0.05). Data presented in C and D were analyzed with ANOVA and Student-Newman-Keuls test. This figure appeared in Chakraborty et al. (2016).

  7. In vivo transfer of virally-manipulated blastocysts to pseudopregnant rats
    1. Pseudopregnant rats are prepared by mating cycling female rats to vasectomized male rats in wire bottom cages. Mating is verified by the identification of seminal plugs beneath the cage. Detection of the seminal plugs is enhanced by placing black paper beneath the wire bottom cages. Detection of seminal plugs is considered day 0.5 of pseudopregnancy.
    2. Following the 4-h incubation of blastocysts with lentiviral vectors and subsequent washes (as described above), then the virally-manipulated blastocysts are transferred to uteri of day 3.5 pseudopregnant rats (~8 blastocysts per uterine horn).
    3. Pregnancies can then be terminated at desired times during gestation and placentation sites interrogated using histological and biochemical approaches (Ain et al., 2006).

Data analysis

For data processing and analyses, please refer to Chakraborty et al. (2016).

Recipes

  1. Outgrowth culture medium
    RPMI 1640
    20% fetal bovine serum
    100 µM 2-mercaptoethanol
    1 mM sodium pyruvate
    50 µM penicillin, and 50 U/ml streptomycin

Acknowledgments

This protocol was adapted from Lee et al. (2009) and Chakraborty et al. (2016). Funding for this work was provided by the NIH, HD020676 and HD079363. Authors declare no conflict of interest or competing interests.

References

  1. Ain, R., Canham, L. N. and Soares, M. J. (2003). Gestation stage-dependent intrauterine trophoblast cell invasion in the rat and mouse: novel endocrine phenotype and regulation. Dev Biol 260(1): 176-190.
  2. Ain, R., Konno, T., Canham, L. N. and Soares, M. J. (2006). Phenotypic analysis of the rat placenta. Methods Mol Med 121: 295-313.
  3. Burton, G. J., Fowden, A. L. and Thornburg, K. L. (2016). Placental origins of chronic disease. Physiol Rev 96(4): 1509-1565.
  4. Chakraborty, D., Cui, W., Rosario, G. X., Scott, R. L., Dhakal, P., Renaud, S. J., Tachibana, M., Rumi, M. A., Mason, C. W., Krieg, A. J. and Soares, M. J. (2016). HIF-KDM3A-MMP12 regulatory circuit ensures trophoblast plasticity and placental adaptations to hypoxia. Proc Natl Acad Sci U S A 113(46): E7212-E7221.
  5. Chiu, S. Y., Maruyama, E. O. and Hsu, W. (2010). Derivation of mouse trophoblast stem cells from blastocysts. J Vis Exp 8(40).
  6. Georgiades, P., Cox, B., Gertsenstein, M., Chawengsaksophak, K. and Rossant, J. (2007). Trophoblast-specific gene manipulation using lentivirus-based vectors. Biotechniques 42(3): 317-318, 320, 322-325.
  7. Georgiades, P., Ferguson-Smith, A. C. and Burton, G. J. (2002). Comparative developmental anatomy of the murine and human definitive placentae. Placenta 23(1): 3-19.
  8. Kent, L. N., Rumi, M. A., Kubota, K., Lee, D. S. and Soares, M. J. (2011). FOSL1 is integral to establishing the maternal-fetal interface. Mol Cell Biol 31(23): 4801-4813.
  9. Knipp, G. T., Audus, K. L. and Soares, M. J. (1999). Nutrient transport across the placenta. Adv Drug Deliv Rev 38(1): 41-58.
  10. Lee, D. S., Rumi, M. A., Konno, T. and Soares, M. J. (2009). In vivo genetic manipulation of the rat trophoblast cell lineage using lentiviral vector delivery. Genesis 47(7): 433-439.
  11. Malashicheva, A., Kanzler, B., Tolkunova, E., Trono, D. and Tomilin, A. (2007). Lentivirus as a tool for lineage-specific gene manipulations. Genesis 45(7): 456-459.
  12. Maltepe, E. and Fisher, S. J. (2015). Placenta: the forgotten organ. Annu Rev Cell Dev Biol 31: 523-552.
  13. Morioka, Y., Isotani, A., Oshima, R. G., Okabe, M. and Ikawa, M. (2009). Placenta-specific gene activation and inactivation using integrase-defective lentiviral vectors with the Cre/LoxP system. Genesis 47(12): 793-798.
  14. Muto, M., Fujihara, Y., Tobita, T., Kiyozumi, D. and Ikawa, M. (2016). Lentiviral vector-mediated complementation restored fetal viability but not placental hyperplasia in Plac1- deficient mice. Biol Reprod 94: 6.
  15. Okada, Y., Ueshin, Y., Isotani, A., Saito-Fujita, T., Nakashima, H., Kimura, K., Mizoguchi, A., Oh-Hora, M., Mori, Y., Ogata, M., Oshima, R. G., Okabe, M. and Ikawa, M. (2007). Complementation of placental defects and embryonic lethality by trophoblast-specific lentiviral gene transfer. Nat Biotechnol 25(2): 233-237.
  16. Pijnenborg, R. and Vercruysse, L. (2010). Chap. 13: Animal models of deep trophoblast invasion. In: Pijnenborg, R., Brosens, I. and Romero, R. (Eds.). Placental bed disorders: basic science and its translation to obstetrics. Cambridge University Press pp: 127-139.
  17. Soares, M. J., Chakraborty, D., Karim Rumi, M. A., Konno, T. and Renaud, S. J. (2012). Rat placentation: an experimental model for investigating the hemochorial maternal-fetal interface. Placenta 33(4): 233-243.
  18. Soares, M. J., Chakraborty, D., Kubota, K., Renaud, S. J. and Rumi, M. A. (2014). Adaptive mechanisms controlling uterine spiral artery remodeling during the establishment of pregnancy. Int J Dev Biol 58(2-4): 247-259.
  19. Watson, E. D. and Cross, J. C. (2005). Development of structures and transport functions in the mouse placenta. Physiology (Bethesda) 20: 180-193.

简介

在这个协议报告中,我们描述了一种慢病毒基因传递技术的基因修改大鼠滋养细胞谱系。 慢病毒包装的基因构建体可以有效地和特异性地递送至胚泡的滋养层细胞谱系。 “功能获得”和“功能丧失”囊胚操作的后果可以用体外生长测定或转移到假孕大鼠后进行评估。

【背景】胎盘作为母亲和发育中的胎儿之间的通道,对于胎儿生殖是必不可少的(Georgiades et。,<2002>)。它是专门用于促进和协调母体对妊娠和胎儿发育的适应性(Soares等人,2014; Burton等人,2016)。胎盘含有滋养层细胞,它们执行多种特殊功能。滋养层细胞特化的获得需要高度调控的滋养层干细胞和祖细胞群的分化(Maltepe and Fisher,2015)。成熟的大鼠胎盘由两个形态上和功能上不同的区室组成:交界区和迷宫区(Soares等人,2012)。祖细胞,海绵状滋养层细胞,糖原滋养层细胞和多倍体滋养层巨细胞构成交界区。侵入性滋养层谱系来自交界区内的祖细胞(Ain等人,2003; Soares等人,2014)。在妊娠的最后一周,这些细胞移出胎盘并侵入母体子宫隔膜室(Ain等人,2003; Pijnenborg和Vercruysse,2010)。胎盘的最内层(接近发育中的胎儿)被称为迷宫区,其由滋养层细胞和来自尿囊的胎儿血管组成。在迷宫区内的祖细胞滋养层细胞融合形成合胞体,这在母体和胎儿隔室之间提供了屏障(Soares等人,2012)。迷宫区由精细的分支结构构成,为胎儿提供了营养,废物和气体交换的大表面积(Knipp等人,1999; Watson和Cross,2005)。

滋养层细胞谱系的特异性修饰可以通过在囊胚阶段使用胚胎外层的慢病毒转导实现,称为滋养外胚层(Georgiades等人,2007; Malashicheva等人, 2007; Okada等人,2007; Lee等人,2009)(图1)。这种方法可以有效地处理所有的滋养层细胞谱系。这也有助于区分滋养层和胚胎对全球基因操作引起的表型结果的贡献。选择性滋养层病毒感染的实验基础与囊胚的结构和由外滋养层形成的紧密上皮直接相关,这有效地限制了病毒颗粒进入内细胞团(Georgiades等人 ,2007; Malashicheva等人,2007; Okada等人,2007; Lee等人,2009)。这种方法可以用来调查滋养细胞谱系的“功能获得”和“功能丧失”操纵的后果,这可以提供对控制胎盘发育的分子机制的见解(Georgiades等人, 2007; Malashicheva等,2007; Okada等,2007; Lee等人,2009; Morioka, 2009; Kent等人,2011; Chakraborty等人,2016; Muto等人, 2016年)。

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图1.显示用于慢病毒转染囊胚和下游分析的实验计划的示意图囊胚用慢病毒颗粒(以绿色显示)转导,表达特定的“功能获得性”或“功能丧失'构建物并培养72小时以进行生长测定或直接转移到3.5天假孕动物体内进行分析。

关键字:囊胚, 胎盘, 滋养层, 慢病毒载体

材料和试剂

  1. 多孔玻璃凝集板(Scientific Device Laboratory,目录号:074-3032B)
  2. 组织培养板,6孔(MIDSCI,目录号:TP92006)
  3. 18针(BD,BD Biosciences,目录号:305195)
  4. 注射器,1毫升
  5. 组织培养板,12孔(MIDSCI,目录号:TP92012)
  6. 玻璃口吸管(Drummond Scientific,目录号:2-000-100)
  7. 吸气管组件,15“(Drummond Scientific,目录号:2-000-000)
  8. 组织培养板,4孔(Thermo Fisher Scientific,Thermo Scientific TM,目录号:176740)
  9. 塑料非组织培养培养皿(Fisher Scientific,目录号:AS4052)
  10. 雌性大鼠(8-10周龄)
  11. 慢病毒包装试剂:pRSV-Rev(Addgene,目录号:12253),pMDLg / pRRE(Addgene,目录号:12251),VSVG包膜质粒(pMD2.G,Addgene,目录号:12259)
  12. 无菌生理盐水溶液(0.9%氯化钠)
  13. Opti-MEM培养基(Thermo Fisher Scientific,目录号:51985034)
  14. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM,目录号:11668-027)
  15. p24酶联免疫测定试剂盒(Takara Bio,Clontech,目录号:632200)
  16. M2培养基(Millipore Sigma,目录编号:MR-015-D)
  17. KSOM培养基(Millipore Sigma,目录号:MR-121-D)
  18. 酸性Tyrode's溶液(Sigma-Aldrich,目录号:T1788)
  19. 矿物油(Sigma-Aldrich,目录号:M8410)
  20. 4%多聚甲醛(Sigma-Aldrich,目录号:158127)
  21. RPMI 1640(Thermo Fisher Scientific,Gibco TM,目录号:11875093)

  22. 胎牛血清,热灭活(西格玛奥德里奇,目录号:F2442)
  23. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  24. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  25. 青霉素和链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)。
  26. (可选)Clontech LentiX滴定试剂盒(Takara Bio,Clontech,目录号:631235)
  27. 生长培养基(见食谱)

设备

  1. 离心机(Beckman Coulter,型号:Optima TM L-100XP,目录号:392050)
  2. 标准倒置光学显微镜或立体显微镜
  3. 标准CO 2细胞培养箱,5%CO 2,37℃。

软件

  1. ImageJ软件(NIH)

程序

  1. 定时妊娠大鼠的制备
    1. 从06:00至20:00(14小时光照:10小时黑暗周期),将动物饲养在环境可控的设施内,并可以自由饮用食物和水。
    2. 雌性大鼠(8-10周龄)与同一品系的雄性大鼠(> 3个月大)放置。
    3. 确认交配是通过检查阴道灌洗来确定的。塑料移液器装有无菌盐水溶液。盐水被送到阴道。将阴道灌洗液转移到干净的多孔玻璃板上并在显微镜下观察。在阴道灌洗中存在精子被认为是妊娠日(gd)0.5。
    注意:
    1. 在大鼠中,阴茎塞保留不常见。因此,有必要获得阴道灌洗,而不是视觉识别精液塞。
    2. 应在08:00-10:00之间进行淋洗以确认精子。如果晚些时候收集,精液可能不容易在阴道灌洗中可见,并可能导致假阴性。















  2. 病毒生产和集中

    1. 293FT细胞在70-80%汇合时分成6孔板
    2. 电镀16-20小时后,将培养基更换为每孔1.5ml的Opti-MEM培养基。

    3. 在培养基更换后继续转染1小时
    4. 在瞬时转染转导载体和第三代包装系统质粒后产生慢病毒。按照生产商的方案使用Lipofectamine 2000制备转染混合物。加入500ng转导载体(shRNA构建体或过表达构建体),200ng pRSV-Rev(Addgene质粒12253),500ng pMDLg / pRRE(Addgene质粒12251)和300ng VSVG包膜质粒(pMD2.G, Addgene质粒12259)。
    5. 8小时后除去转染混合物,加入2ml补充有5%胎牛血清的Opti-MEM培养基。
    6. 20小时后取出培养基并收集在储存在4℃的试管中。
      两个集合在40-48小时内执行
    7. 离心培养物上清液以除去细胞碎片,过滤灭菌并通过超速离心(35,000xg克,3小时)浓缩。储存在-80°C直到用于转导。不要冻融。
    8. 慢病毒载体滴度应通过酶联免疫分析法测定p24 gag抗原来确定。

  3. 胚胎收集

    1. 在08:30-09:30之间收集gd 4.5怀孕大鼠的生殖道
    2. 子宫从脂肪组织中分离出来,并通过在子宫 - 输卵管连接处和子宫颈处切割来切除。
    3. 用子宫颈末端的M2介质冲洗尿道,用18号针头连接1ml注射器。
    4. 冲洗的渗出液收集在12孔组织培养板的一个孔中。
    5. 用口吸取器回收囊胚并将其转移到一滴KSOM培养基中。
    6. 囊胚在CO 2培养箱中于37℃保持30分钟以稳定处理相关的压力。
    注意:
    1. 采集时间对于获得囊胚阶段胚胎是非常重要的。如果过早采集,胚胎将在输卵管中,冲洗子宫不会获得胚泡阶段的胚胎。如果收集太晚,胚胎开始附着在子宫上,通过冲洗子宫很难取回胚胎。
    2. 应立即将胚胎转移到37℃的5%CO 2培养箱中的KSOM培养基中,不要留在未缓冲的培养基中延长期限。
    3. 有关胚胎冲洗和收集的详细信息,请参阅Chiu等人,2010。

  4. 从囊胚中去除透明带

    1. 在培养皿中放置两个独立的Acid Tyrode溶液液滴(约50μl液滴尺寸)
    2. 在第二个培养皿中,准备5个独立的KSOM培养基液滴(大约40μl液滴大小)。
    3. 在倒置显微镜下,将囊胚转移至第一个酸性Tyrode溶液液滴并监测透明带的完整性。如果透明带溶解不完全,则将囊胚移入第二滴,然后立即将它们转移至KSOM中滴。
    4. 在成功去除透明带和5次KSOM洗涤之后,将囊胚转移到覆盖有矿物油的KSOM液滴中。矿物油应完全覆盖KSOM液滴,所需矿物油的总体积将取决于培养皿的大小(2-3毫升将足以覆盖KSOM液滴)。
    5. 在37°C孵育胚泡进一步稳定。
    注意:
    1. 胚胎的存活依赖于孵育时间和暴露于酸性Tyrode溶液。过度暴露于酸性Tyrode溶液导致胚胎破裂和塌陷,这将不适合生长或体内转移实验。
    2. 当胚胎转移到酸性滴定溶液时,当透明带开始离解时,必须经常观察以立即将其移除。一旦透明带被移除,胚胎应立即清洗并转移到KSOM液滴。
    3. 去除透明带后,胚胎变得粘稠并倾向于聚集。








  5. 病毒孵化和洗涤
    1. 如前所述(Lee等人,2009),产生含有“功能获得性”或“功能丧失”构建体的慢病毒颗粒,浓缩并保存。
    2. 在30微升KSOM液滴中加入10微升浓缩的病毒。
    3. 将囊胚转移到含有病毒颗粒的KSOM液滴中。液滴应覆盖矿物油(2-3毫升将足以覆盖KSOM液滴)。

    4. 在37°C孵育病毒囊胚4小时。
    5. 孵化后,依次用10-KSOM液滴洗囊胚。
    6. 第10次洗涤后,将囊胚转移到被矿物油覆盖的KSOM液滴中,并在37℃下孵育15分钟。
    注意:
    1. p24外壳蛋白测量给出病毒浓度的相对评估,并不一定表明病毒批次的感染性的功效。
    2. 可选的qPCR方法(Clontech LentiX滴定试剂盒)也可用于测量病毒滴度。 1 x 10 8 至3 x 10 8 感染单位/毫升可用于孵化囊胚的有效转导。
    3. 500ng p24 / ml的病毒浓度对于转导是有效的。但是,有效的病毒颗粒浓度需要根据每个实验应用凭经验确定。

  6. 准备囊胚生长
    1. 在4孔组织培养板的单个孔中加入750μl生长培养基(见配方)。

    2. 每个孔加入一个病毒操纵的囊胚,并在37°C孵育
    3. 每天观察胚泡,直到囊胚附着在4孔组织培养板上。
    4. 一旦囊胚连接,删除介质,并添加新鲜的媒介。通常情况下,胚泡需要2-3天的时间来孵化和附着。
    5. 在孵化的第5天,在对照囊胚中会观察到生长物(图2)。
    6. 产物可以固定在4%多聚甲醛中用于免疫荧光,或者可以收获用于进一步的生物化学/分子分析。


      图2.氧张力和KDM3A表达对胚泡生长的影响A.示意图显示了用于囊胚的慢病毒转导和生长测定的实验计划。将囊胚用对照(Ctrl)或Kdm3a shRNA转导并培养72小时以允许来自透明带的孵化。将附着的囊胚暴露于环境(Amb)或低氧(0.5%O 2)24小时并分析。 B.代表性的从Ctrl shRNA中暴露于囊胚并暴露于Amb,Ctrl shRNA并暴露于0.5%O 2和Kdm3a shRNA并暴露于0.5%O 2的胚泡的图像。 C.通过qRT-PCR测量对照和敲低培养物中Kdm3a转录物的测量。星号表示组间差异显着(n = 6 /组; P <0.05)。 D.条形图以平方毫米显示生长区域的量化。使用ImageJ软件(Ctrl shRNA + Amb,n = 6; Ctrl shRNA + 0.5%O 2,Kdm3a shRNA1 + 0.5%O 2)测量生长的面积, n = 10; Kdm3a shRNA2 + 0.5%O 2,n = 10; * <0.05)。用C和D表示的数据用ANOVA和Student-Newman-Keuls试验进行分析。这个数字出现在 Chakraborty pnas.org/content/113/46/E7212.full.pdf“target =”_ blank“>(2016)
  7. 将病毒处理的胚泡体内转移至假孕大鼠
    1. 假孕大鼠是通过将雌性大鼠交配到铁丝网底笼中的输精管结扎的雄性大鼠而制备的。通过在笼子下方的精液塞的鉴定来验证交配。在导线底部笼子下面放置黑色的纸张可以增强对精液塞的检测。
      。精囊炎的检测被认为是假孕的第0.5天
    2. 在囊胚与慢病毒载体孵育4小时并随后洗涤(如上所述)之后,然后将病毒操纵的囊胚转移到3.5天假孕大鼠(〜8个囊胚/子宫角)的子宫中。
    3. 怀孕可以在妊娠期和胎盘部位使用组织学和生物化学方法询问期望的时间终止(Ain et al。2006)。

数据分析

有关数据处理和分析,请参阅 Chakraborty (2016)

食谱

  1. 生长培养基
    RPMI 1640
    20%胎牛血清
    100μM2-巯基乙醇
    1 mM丙酮酸钠
    50μM青霉素和50 U / ml链霉素

致谢

该协议改编自Lee等人(2009)和Chakraborty等人。 (2016)。这项工作的资金由NIH提供,HD020676和HD079363。作者声明不存在利益冲突或利益冲突。

参考

  1. Ain,R.,Canham,L.N。和Soares,M.J。(2003)。妊娠期依赖于宫内滋养层细胞侵袭的大鼠和小鼠:新型内分泌表型和调节 / 1> Dev Biol 260(1):176-190。
  2. Ain,R.,Konno,T.,Canham,L.N。和Soares,M.J。(2006)。 大鼠胎盘的表型分析 Methods Mol Med
    121:295-313
  3. Burton,G. J.,Fowden,A. L.和Thornburg,K. L.(2016)。 慢性疾病的胎盘起源 Physiol Rev 96( 4):1509-1565。
  4. Chakraborty,D.,Cui,W.,Rosario,GX,Scott,RL,Dhakal,P.,Renaud,SJ,Tachibana,M.,Rumi,MA,Mason,CW,Krieg,AJ和Soares,MJ(2016) 。 HIF-KDM3A-MMP12调控回路确保滋养层可塑性和胎盘适应缺氧。 (Proc Natl Acad Sci USA)113(46):E7212-E7221。
  5. Chiu,S.Y.,Maruyama,E.O.和Hsu,W。(2010)。 从胚泡衍生小鼠滋养层干细胞 J Vis Exp 8(40)。
  6. Georgiades,P.,Cox,B.,Gertsenstein,M.,Chawengsakophaophak,K.和Rossant,J。(2007)。 使用基于慢病毒载体的滋养层特异性基因操作生物技术 (3):317-318,320,322-325。
  7. Georgiades,P.,Ferguson-Smith,A.C和Burton,G.J。(2002)。 小鼠和人类胎盘的比较发育解剖学胎盘23(1):3-19。
  8. Kent,L.N.,Rumi,M.A.,Kubota,K.,Lee,D.S。和Soares,M.J。(2011)。 FOSL1是建立母胎界面不可或缺的部分。 分子细胞生物学 31(23):4801-4813。
  9. Knipp,G.T.,Audus,K.L。和Soares,M.J。(1999)。 营养素通过胎盘运输。 高级药物递送 38(1):41-58。
  10. Lee,D.S。,Rumi,M.A.,Konno,T。和Soares,M.J。(2009)。使用慢病毒载体递送对大鼠滋养层细胞谱系进行体内基因操作( 。创世纪 47(7):433-439。
  11. Malashicheva,A.,Kanzler,B.,Tolkunova,E.,Trono,D。和Tomilin,A。(2007)。 慢病毒作为谱系特异性基因操作的工具。 45(7):456-459。
  12. Maltepe,E.和Fisher,S.J。(2015)。 胎盘:被遗忘的器官 Annu Rev Cell Dev Biol 31:523-552。
  13. Morioka,Y.,Isotani,A.,Oshima,R.G.,Okabe,M。和Ikawa,M。(2009)。 使用Cre / LoxP系统使用整合酶缺陷型慢病毒载体的胎盘特异性基因激活和失活< />创世纪 47(12):793-798。
  14. Muto,M.,Fujihara,Y.,Tobita,T.,Kiyozumi,D。和Ikawa,M。(2016)。 慢病毒载体介导的互补在Plac1缺陷型小鼠中恢复了胎儿的存活力,但不是胎盘增生。
  15. Okada,Y.,Ueshin,Y.,Isotani,A.,Saito-Fujita,T.,Nakashima,H.,Kimura,K.,Mizoguchi,A.,Oh-Hora,M.,Mori,Y.,Ogata M.,Oshima,RG,Okabe,M。和Ikawa,M。(2007)。 滋养细胞特异性慢病毒基因转移补充胎盘缺陷和胚胎致死率 Nat Biotechnol 25(2):233-237。
  16. Pijnenborg,R.和Vercruysse,L.(2010)。 Chap。 13:深滋养细胞侵袭的动物模型。在:Pijnenborg,R.,Brosens,I。和Romero,R。(编辑)。胎盘床障碍:基础科学及其产科翻译。剑桥大学出版社 pp:127-139。
  17. Soares,M.J.,Chakraborty,D.,Karim Rumi,M.A.,Konno,T。和Renaud,S.J。(2012)。 大鼠胎盘素:一种调查血红素母胎界面的实验模型 胎盘 33(4):233-243。
  18. Soares,M.J.,Chakraborty,D.,Kubota,K.,Renaud,S.J。和Rumi,M.A。(2014)。 在妊娠期间控制子宫螺旋动脉重塑的适应性机制 Int J Dev Biol 58(2-4):247-259。
  19. Watson,E.D。和Cross,J.C。(2005)。 小鼠胎盘结构和运输功能的发展生理学(Bethesda ) 20:180-193。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chakraborty, D., Muto, M. and Soares, M. J. (2017). Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery. Bio-protocol 7(24): e2652. DOI: 10.21769/BioProtoc.2652.
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