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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Streszczenie

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Wprowadzenie

Genetic manipulation of the embryonic mouse brain is a preferred approach to learn about developmental regulation. The generation of mutant mouse lines however is slow and expensive. One powerful method to introduce specific genetic changes in developing neurons of the mammalian brain is in utero electroporation. Essentially, the technique consists of transfecting DNA into the embryonic brain neuroepithelium by means of electric pulses, then allowing the embryo to survive for a certain period of time, collect the brain and examine them for possible novel, informative phenotypes. In this way, the experimenter can test hypotheses almost immediately without the long waiting periods necessary for the production of mouse mutants.

Transfection of DNA into developing embryos started with in ovo electroporation on chick embryos1. The essential proof-of-concept for the mouse was performed in culture2. This was soon followed by the first descriptions of the technique on the mouse in utero3,4.

The main problem is to transfect the brain of embryos developing in utero without killing them or the mother. Learning to perform the necessary surgery (laparotomy, injection, electroporation) requires a long training period. Once the surgery has been mastered to the point where the embryo survival ratio is acceptable, the next key question is: which brain structures are accessible? Not surprisingly, the first published papers showing results obtained with in utero electroporation focused on cortical development5-9. This is still true for most of the publications using this technique, since the region of the developing mouse brain most accessible to surgical procedures is the cortex (Figure 1). The procedure for in utero electroporation into the cortex has been described in print10 and in video11-14. A modification of the technique can be used to target a ventral part of the telencephalon, the basal ganglia15.

Beyond the telencephalon, the diencephalon (classically divided into thalamus and hypothalamus) is a region of the forebrain more difficult to reach. A small number of papers reports targeting of its dorsal and most accessible part, the thalamus16-19.

The hypothalamus is the most ventral part of the forebrain, therefore the one localized most deeply from the dorsal surface (cortex) (Figure 1). This region remains a difficult challenge for researchers committed to genetically manipulate the mouse brain in utero. To our knowledge, only very few articles report on results of in utero transfection into the mouse hypothalamus 20,21. However, the functional importance of the hypothalamus cannot be overstated, since it regulates behaviors like eating and drinking, mating, breeding and parenting22. Moreover, alterations in hypothalamic development contribute to originate later in life conditions like obesity, hypertension, diabetes and precocious puberty23. Being able to alter genetically the hypothalamus during development would provide a very powerful tool to understand it.

The basic surgical protocol for the laparotomy of pregnant mice that we use here is similar to that used in other protocols11,13,14,24. We will describe them here briefly for completeness. Key to our procedure, on the other hand, are the type of anesthesia, the place of injection, the type of electrodes and the insertion and position of the positive electrode with respect to the embryo's head. We prefer to induce and maintain anesthesia through gas inhalation over simple intraperitoneal anesthesia, since the former allows for the somewhat longer periods of narcosis required for a difficult surgery. Isoflurane inhalation results in faster recovery from anesthesia, since usually the mother demonstrates normal behavior already minutes after the surgery. The easiest point of injection of the DNA solution with the glass micropipette is the lateral ventricle, which however is completely unsuitable for hypothalamus electroporation. Injection directly in the third ventricle is indeed crucial to target deep diencephalic structures. It is possible to transfect the hypothalamus from E12.0 or E12.5 with standard, off-the-shelf electrodes. We have found some of the electrodes manufactured by Nepa Gene (Chiba, Japan) particularly suited to this purpose.

With our procedure we obtain transfection of the entire hypothalamic neuroepithelium or partial, regional transfection depending on electrode orientation. Here we demonstrate the technique by transfecting the mammillary body, arguably the deepest and most recessed of all hypothalamic nuclei. Additionally, we show detailed histological analysis of the transfected cells down to the cellular level of resolution.

A comparison of in utero electroporation with other approaches to transfecting the mouse developing brain in utero can be found in the Discussion section.

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Protokół

1. Preparation of DNA and Glass Micropipettes for Injection

  1. Good quality glass micropipettes are essential to reduce initial high abortion rate due to loss of amniotic fluid. The procedure to pull glass micropipettes has been well documented13,18,25. Use 1.2 mm diameter capillaries pulled in a conventional Sutter P-97 device with the settings P=500; Heat=300; Pull=40; Velocity=50; Time=50. Fit the puller with 3 mm "trough" filaments (Sutter Instrument FT330B). The 2 mm size filaments have yielded for us less satisfactory results. On the other hand, beveling of the micropipette tips does not seem to improve results for us.
  2. Dissolve purified, endotoxin-free plasmid DNA in PBS (Cell Culture Grade) containing 0.1% Fast Green to a final concentration of 1 to 2 μg/μl. The Fast Green will make the injected solution visible in the embryonic brain ventricle.
  3. Load 10 μl of the DNA solution into the glass micropipette.
  4. Connect the glass micropipette to the injection system (Pico Pump) or mouth pipette.

2. Anesthesia

Prepare the surgical table with a heating pad and the surgical instruments. Turn on the cold-light sources to facilitate the visualization of the embryos. Disinfect the surgical tools using for instance a glass bead sterilizer.

Three different anesthesia procedures are possible for this protocol (see Discussion). Here we will describe the one we consider the best, using isoflurane inhalation for anesthesia induction (flow rate 0.5 L/min) as well as maintenance (flow rate 1 L/min).

  1. Introduce the pregnant mouse in a small transparent (so the mouse remains visible) container connected to the Komesaroff Mark 5 Anesthetic Device by a short length of tubing.
  2. Fill the vaporizer with isoflurane, then open the oxygen bottle attached to the device. Blow one or two pulses of isoflurane/oxygen (0.5 L/min) into the container holding the mouse and watch for narcosis to set in.
  3. Take the mouse out, cover its eyes with ointment to prevent them from drying and fit the flow anesthesia mask on its head immediately.

3. Laparotomy

  1. Place the mouse belly up on the heating pad (otherwise its body temperature will go down very fast, decreasing chances of recovery) and secure its body into place by fixing its four legs to the sides with tape. Assess the depth of anesthesia by checking for loss of response to reflex stimulation (e.g. toe or tail pinch with firm pressure).
  2. Shave the abdominal surface and disinfect it with the iodophorpovidone-iodine (Braunoderm).
  3. Make a longitudinal incision (1 to 1.5 cm long) on the abdominal skin. Then cut the peritoneum. Place cotton gauze around the incision. Make one uterine horn visible and pull it out carefully with blunt forceps onto the PBS-rinsed gauze. Rinse the uterus with PBS very often to keep it always moistened (Figures 2A and 2B). During the entire procedure avoid pulling the mesometrium or the uterus tight, since high pressure inside the uterus will transmit to the embryo increasing the chances of loss of fluid upon injection resulting in abortion.

4. DNA Injection into the Brain Ventricle

  1. Hold the uterus in such a way that the brain ventricles can be visualized. Do not extensively reposition an embryo that lies in an unfavorable position - this only increases the chances of abortion.
  2. Looking at the embryo's head from top, localize visually the gap or fissure between the left and right cortical hemispheres. The hemispheres are easy to distinguish and the lateral ventricles inside them (not to be targeted) can usually be perceived as somewhat darker shapes. If an embryo is found to be perfectly oriented for DNA injection (Figures 2C and 2D), it is possible to pierce the uterine wall and enter the third ventricle at once. Hold the glass micropipette at 45° to the uterine wall and puncture it at the rostral end of the gap between cortical hemispheres, penetrating for about 1 mm. In this way the tip of the glass micropipette will enter the third ventricle of the brain (not the lateral ventricle) (Figures 2C and 2D). In embryos less favorably oriented it is useful to first pierce the uterine wall (always at 45°) in the vicinity of the embryonic head, place the micropipette tip in the right position between the cortical hemispheres and only then perforate the brain. Inject about 1 μl of DNA solution into the third ventricle (a good injection fills the ventricle with green fluid). Repeat the same procedure with all embryos of one uterus horn. This allows some time for the DNA solution to mix evenly with the ventricular fluid and reach the entire neuroepithelium.

5. Targeting the Hypothalamus for Electroporation

  1. Switch the electroporator on and adjust settings according to the embryonic age (for E12.5 we use 5 square-wave pulses, 50 V, 50 msec ON/950 msec OFF). Use as positive pole the stainless steel needle electrode (CUY550-10) and as negative pole a round flat electrode (CUY700P4L). (It is important that the electrodes are smaller than the embryo, since otherwise the current just flows around the embryo but not through it.)
  2. Select for electroporation those embryos whose dorsal side is up (i.e. turned towards the experimenter) (as most of them are), and discard those whose orientation is not favorable. It is now safe to touch the uterus with ethanol-disinfected fingers or with gloves. Holding the uterus with forceps, however, would interfere with the flow of current during electroporation.
  3. Pierce the uterine wall by the embryo's head between the amniotic sac and the placenta by thrusting downwards through it with the tip of the needle electrode. Use the index finger of the other hand as thrust block. About 5 mm of the electrode tip must be now between the amniotic sac and the uterine wall, at about the level of the midbrain (Figures 2E and 2F). Remember that this is the "targeting electrode", so its position will determine which part of the hypothalamic neuroepithelium is most likely to get transfected. The embryo has to be very gently "squeezed" between the electrodes.
  4. With the other hand, position the round flat electrode outside of the uterus wall on the opposite side of the embryo's head (Figures 2E and 2G).
  5. Use the pedal switch to apply voltage (50 V, 50 msec ON, 950 msec OFF, 5 square-wave pulses).
  6. Slowly pull the needle electrode out of the uterus while holding back the uterus with the index finger of the other hand - if amniotic liquid is lost through the punctured wall, usually the embryo will undergo abortion. Repeat the procedure with all the embryos.
  7. Return the uterus horn into the abdomen and repeat the injection and electroporation in the remnant horn.

6. Finishing the Surgery

  1. After injection and electroporation of all embryos, place the uterus back into the abdomen very carefully, positioned exactly as they were before and moist the peritoneal cavity with saline before closing it.
  2. Suture the peritoneum with surgical catgut (Vicryl Polyglactin 910, 5-0, Ethicon V4914). For the closure of the skin use "interrupted stitch" methodwith a more resistant suture (Supramid Nylon, 6-0, Serag Wiessner TO 07171L).
  3. Disinfect the abdomen surface with povidone-iodine(Braunoderm) and inject a non-steroid anti-inflammatory subcutaneously (e.g. 100 μl of a 1:10 solution of Rimadyl in 0.9% NaCl) or, even better, an opioid like buprenorphine (Temgesic, 0.05 to 0.1 mg/kg body weight in 0.9% NaCl)to relieve pain.
  4. Remove the mother from the anesthetic machine and place it in a cage which is heated by a heating pad. Monitor the mouse continually until it is completely recovered from the anesthesia. Later on, check the animals daily to insure they are recovering from the procedure without any sign of infection or pain.

7. Analyzing the Results

  1. Harvest the embryos (or postnatals) according to the desired day of analysis. Postnatal mice (1 to 2 day old) are killed by decapitation.
  2. Separate every embryo according to the previous electroporation annotation. Dissect the brain under a stereomicroscope and check under the microscope for green fluorescent signal (from the GFP reporter) in the appropriate region.
  3. The selected brain can be analyzed "fresh": fix the tissue for a short time in 4% paraformaldehyde and embed in agarose 4% or in gelatin-albumin, then section on a vibratome-type device (Figure 3). For more detailed immunohistochemical analysis (Figure 4), cryo-protect the brain in 30% saccharose solution, embed in OCT mounting medium (Tissue Tek) then cut 20 μm sections in a cryostat.

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Wyniki

Most hypothalamus neurons are born between E11.5 to E15.2, according to birth-dating analysis in the rat26 translated into the somewhat shorter mouse development27,28. The peak of hypothalamic neurogenesis is reached at E12.529-31. Accordingly, at the transfection age chosen for the present study (E12.5), a large proportion of hypothalamic neurons can be labeled at any given rostro-caudal level.

Analysis at E18.5 on thick vibratome-type sections (Figur...

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Dyskusje

About the anesthesia: Since in utero electroporation into the hypothalamus can be technically arduous and require longer narcosis times, we prefer to induce and maintain anesthesia through administration of a mixture of oxygen and isoflurane. In our experience, animals can remain suitably anaesthetized in this way for periods of up to one hour at least, the recovery of the mother is very fast, and embryo survival improved. Other approaches to anesthesia are also available. The most simple procedure consis...

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Ujawnienia

The authors declare that they have no competing financial interests.

Podziękowania

This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft).

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Materiały

NameCompanyCatalog NumberComments
 REAGENTS
AcepromazineSanofi GmbHanesthetic
IsofluraneBaxterHDG9623anesthetic
KetaminPharma GmbHanesthetic
Fast GreenFluka44715 
RimadylPfizernon-steroidal anti-inflammatory
BraunodermBraun3887138povidone-iodine
Phosphate Buffer Saline PBSGibco14190 
Temgesic (buprenorphine)Essex Pharmaopioid analgesic
Eye OintmentPan-Ophtal7136926 
XylazineBayer 
 EQUIPMENT
Anaesthetic Device Komesaroff Mark-5Medical Developments AustraliaACN 004 903 682 
Capillary puller P-97Sutter Instrument Co.P-97 
CompresstomePrecisionary Instr.VF-300Vibratome-type device
Confocal MicroscopeZeissLSM700 
CryostatLeicaCM3050S 
ElectroporatorNepa Gene Co. Ltd.CUY21EDIT 
Electrode 1Nepa Gene Co. Ltd.CUY550-10Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.
Electrode 2Nepa Gene Co. Ltd.CUY700P4LCover Round Platinum Plate 4 mm diameter
Fiberoptic cold light sourceLeicaKL2500 LCD 
Glass capillariesHarvard ApparatusGC120T-151. 2 mm O.D. x 0. 94 mm I.D.
Glass bead sterilizerFine Science ToolsFST250 
Heating padHarvard Apparatuspy872-5272 
Injection deviceWorld Precision InstrumentsPneumatic Pico Pump PV820 
Suture Thread Coated VicrylEthiconV4914Peritoneal Suture
Suture Thr. SupramidSerag WiessnerTO07171LSkin Suture

Odniesienia

  1. Itasaki, N., Bel-Vialar, S., Krumlauf, R. Shocking' developments in chick embryology: electroporation and in ovo gene expression. Nat. Cell Biol. 1, E203-E207 (1999).
  2. Miyasaka, N., Arimatsu, Y., Takiguchihayashi, K. Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation. Neuroreport. 10, 2319-2323 (1999).
  3. Saito, T., Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237-246 (2001).
  4. Tabata, H., Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience. 103, 865-872 (2001).
  5. Fukuchi-Shimogori, T., Grove, E. A. Neocortex patterning by the secreted signaling molecule FGF8. Science. 294, 1071-1074 (2001).
  6. Tabata, H., Nakajima, K. Neurons tend to stop migration and differentiate along the cortical internal plexiform zones in the Reelin signal-deficient mice. J. Neurosci. Res. 69, 723-730 (2002).
  7. Fukuchi-Shimogori, T., Grove, E. A. Emx2 patterns the neocortex by regulating FGF positional signaling. Nat. Neurosci. 6, 825-831 (2003).
  8. Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex. Development. 131, 5639-5647 (2004).
  9. Shimogori, T., Grove, E. A. Fibroblast growth factor 8 regulates neocortical guidance of area-specific thalamic innervation. J. Neurosci. 25, 6550-6560 (2005).
  10. Saito, T. In vivo electroporation in the embryonic mouse central nervous system. Nat. Protoc. 1, 1552-1558 (2006).
  11. Dixit, R., et al. Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation. J. Vis. Exp. (52), e2957(2011).
  12. Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons. J. Vis. Exp. (44), e2103(2010).
  13. Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos. J. Vis. Exp. (6), e239(2007).
  14. Walantus, W., Elias, L., Kriegstein, A. In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos. J. Vis. Exp. (6), e236(2007).
  15. Borrell, V., Yoshimura, Y., Callaway, E. M. Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J. Neurosci. Methods. 143, 151-158 (2005).
  16. Haddad-Tovolli, R., Heide, M., Zhou, X., Blaess, S., Alvarez-Bolado, G. Mouse thalamic differentiation: gli-dependent pattern and gli-independent prepattern. Front Neurosci. 6, 27(2012).
  17. Kataoka, A., Shimogori, T. Fgf8 controls regional identity in the developing thalamus. Development. 135, 2873-2881 (2008).
  18. Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection. J. Vis. Exp. (54), e3024(2011).
  19. Vue, T. Y., et al. Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice. J. Neurosci. 29, 4484-4497 (2009).
  20. Tanaka, S., et al. Transcriptional regulation of the hypocretin/orexin gene by NR6A1. Biochem. Biophys. Res. Commun. 403, 178-183 (2010).
  21. Tsuchiya, R., Takahashi, K., Liu, F. C., Takahashi, H. Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections. J. Neurosci. Res. 87, 1620-1633 (2009).
  22. Canteras, N. S. The Mouse Nervous System. Watson, C., Paxinos, G., Puelles, L. , Academic Press. 539-562 (2011).
  23. Caqueret, A., Yang, C., Duplan, S., Boucher, F., Michaud, J. L. Looking for trouble: a search for developmental defects of the hypothalamus. Horm. Res. 64, 222-230 (2005).
  24. Petros, T. J., Rebsam, A., Mason, C. A. In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells. J. Vis. Exp. (31), e1333(2009).
  25. Nijagal, A., Le, T., Wegorzewska, M., Mackenzie, T. C. A Mouse Model of in Utero Transplantation. J. Vis. Exp. (47), e2303(2011).
  26. Altman, J., Bayer, S. A. The Development of the Rat Hypothalamus. Adv. Anat. Embryol. Cell Biol. 100, 1-178 (1986).
  27. Clancy, B., Darlington, R. B., Finlay, B. L. Translating developmental time across mammalian species. Neuroscience. 105, 7-17 (2001).
  28. Clancy, B., et al. Web-based method for translating neurodevelopment from laboratory species to humans. Neuroinformatics. 5, 79-94 (2007).
  29. Ishii, Y., Bouret, S. G. Embryonic birthdate of hypothalamic leptin-activated neurons in mice. Endocrinology. 153, 3657-3667 (2012).
  30. Karim, M. A., Sloper, J. C. Histogenesis of the supraoptic and paraventricular neurosecretory cells of the mouse hypothalamus. J. Anat. 130, 341-347 (1980).
  31. Shimada, M., Nakamura, T. Time of neuron origin in mouse hypothalamic nuclei. Exp. Neurol. 41, 163-173 (1973).
  32. Alvarez-Bolado, G., Paul, F. A., Blaess, S. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions. Neural. Dev. 7, 4(2012).
  33. Vann, S. D., Aggleton, J. P. The mammillary bodies: two memory systems in one. Nat. Rev. Neurosci. 5, 35-44 (2004).
  34. Gratsch, T. E., De Boer, L. S., O'Shea, K. S. RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos. Genesis. 37, 12-17 (2003).
  35. Wu, N., Yu, A. B., Zhu, H. B., Lin, X. K. Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad. J. Biomed. Biotechnol. 2012, 343891(2012).
  36. Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., Sato, M. Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection. Gene Ther. 9, 1529-1541 (2002).
  37. Foust, K. D., et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59-65 (2009).
  38. Xin, H., et al. The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles. Biomaterials. 33, 1673-1681 (2012).
  39. Niwa, H., Yamamura, K., Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199 (1991).
  40. Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res. 19, 4485-4490 (1991).
  41. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509-544 (1998).
  42. Londrigan, S. L., et al. Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets. Xenotransplantation. 14, 119-125 (2007).

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Keywords Genetic ManipulationMouseDeveloping HypothalamusIn Utero ElectroporationGenetic ModificationMammalian BrainMouse MutantsDeveloping BrainCortexThalamusHypothalamusHypothalamic NeuroepitheliumMammillary BodyElectroporation

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