Name: लेखक स्पॉटलाइट: माइक्रोबायोटा-होस्ट रिलेशनशिप स्टडीज के लिए रोगाणु मुक्त ज़ेब्राफिश की स्थापना
Uploaded: 2024-04-12T13:50:00
Description: Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

हम ईमानदारी से चोंगकिंग मेडिकल यूनिवर्सिटी टैलेंट प्रोजेक्ट (नंबर R4014 से DSP और R4020 से PPJ), नेशनल नेचुरल साइंस फाउंडेशन ऑफ चाइना (NSFC, नंबर 32200386 से PPJ), चोंगकिंग पोस्टडॉक्टोरल इनोवेशन मेंटर स्टूडियो (X7928 DSP), और चाइनीज एकेडमी ऑफ साइंसेज (CAS)/चीन-श्रीलंका ज्वाइंट सेंटर फॉर एजुकेशन एंड रिसर्च द्वारा चाइनीज एकेडमी ऑफ साइंसेज (CAS)/चीन-श्रीलंका ज्वाइंट सेंटर फॉर एजुकेशन एंड रिसर्च के कार्यक्रम के समर्थन का धन्यवाद करते हैं।

मछली के प्रयोग चूंगचींग की पशु देखभाल और उपयोग समिति और चोंगकिंग मेडिकल यूनिवर्सिटी, चीन की संस्थागत पशु देखभाल और उपयोग समिति के दिशानिर्देशों के साथ-साथ गुणवत्ता और तकनीकी पर्यवेक्षण राज्य ब्यूरो ...

GF Zebrafish मॉडल का अनुकूलन: जनरेशन, रखरखाव और माइक्रोबियल अध्ययन

GF मछली और GF भोजन तैयार करने के प्रोटोकॉल के भीतर महत्वपूर्ण कदम जीएफ मछली मॉडल की पीढ़ी के दौरान, बाँझ सामग्री की तैयारी, भ्रूण की नसबंदी, GZM के दैनिक नवीकरण, विभिन्न नमूनों के संग्रह, और कई तरीकों क...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AB-GZM</td>
			<td>Amphotericin:Solarbio;&#160; kanamycin:Solarbio; Ampicillin:Solarbio.</td>
			<td>Amphotericin:CAS:1397-89-3; 
			kanamycin:CAS: 25380-94-0; Ampicillin:CAS: 69-52-313.</td>
			<td>49.6 mL GZM, 50 &#181;L amphotericin stock solution (250 &#181;g/mL), 25 &#181;L kanamycin stock solution (10 mg/mL), and 250 &#181;L ampicillin stock solution (20 mg/mL).</td>
		</tr>
		<tr>
			<td>1.5 mL, 15 mL, 50 mL EP tubes</td>
			<td>biosharp</td>
			<td>BS-15-M</td>
			<td>To collect samples, and hold agents</td>
		</tr>
		<tr>
			<td>2.4 g/L NaClO</td>
			<td>XILONG SCIENTIFIC Co., Ltd.</td>
			<td>CAS: 7681-52-9</td>
			<td>Diluted with 8% sodium hypochlorite aqueous solution.</td>
		</tr>
		<tr>
			<td>6-well plates, 24-, 48- well plates</td>
			<td>LABSELECT&#160;</td>
			<td>11112</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>Aeronomas</td>
			<td>NCBI database</td>
			<td>No.MK178499</td>
			<td>2019-JPP-ESN</td>
		</tr>
		<tr>
			<td>Anaerobic TSA plates</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp; 
			agar powder:BioFroxx.</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112; 
			agar powder:9002-18-0.</td>
			<td>The TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, and 6 g agar powder under the anaerobic system.</td>
		</tr>
		<tr>
			<td>Anaerobic work station</td>
			<td>GENE SCIENCE</td>
			<td>E200G</td>
			<td>Bacterial isolation, sterile testing</td>
		</tr>
		<tr>
			<td>Analysis</td>
			<td>GraphPad Prism 5</td>
			<td>v6.07</td>
			<td>To analysis the data</td>
		</tr>
		<tr>
			<td>API 20 E kits</td>
			<td>&#160;BioMerieux SA, France</td>
			<td>No.1005915090</td>
			<td>Ref 20100 Kits to detect bacterial metabolism</td>
		</tr>
		<tr>
			<td>Artemia (Brine shrimp)</td>
			<td>Shangjia Aquarium Co., Ltd.</td>
			<td>Aquamaster brand</td>
			<td>Artemia cysts, and brine shrimp eggs&#160;</td>
		</tr>
		<tr>
			<td>Auto cycle system for fish culture</td>
			<td>Ningbo Hairui Technology Co., Ltd</td>
			<td>No Cat</td>
			<td>Maintain the fish</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Zeal Way</td>
			<td>G154DWS</td>
			<td>Prepare the materials</td>
		</tr>
		<tr>
			<td>BHI Aerobic</td>
			<td>Coolaber</td>
			<td>Cat#PM0640</td>
			<td>BHI medium was prepared, wherein 100 mL medium included 3.7 g BHI powder.</td>
		</tr>
		<tr>
			<td>BHI Anaerobic</td>
			<td>Coolaber</td>
			<td>Cat#PM0640</td>
			<td>BHI medium was prepared and divided into anaerobic tubes under the anaerobic system.</td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>LongYue Co., Ltd</td>
			<td>SPX</td>
			<td>For fish and plates</td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Haier</td>
			<td>HR40-IIA2</td>
			<td>Sterile treatment and testing</td>
		</tr>
		<tr>
			<td>Bleaching agent of 0.02 g/L NaClO</td>
			<td>XILONG SCIENTIFIC Co., Ltd.</td>
			<td>CAS: 7681-52-9</td>
			<td>Working solution with sodium hypochlorite (NaClO) concentration: Diluted with 8% sodium hypochlorite aqueous solution or 166.6 uL 6% sodium hypochlorite with 500 mL distilled water.</td>
		</tr>
		<tr>
			<td>Blood plates</td>
			<td>sheep blood:Solarbio</td>
			<td>Cat. NO. TX0030</td>
			<td>Sterile-defibrinated sheep blood was added into TSA to prepare 5% blood plates.</td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>CM-Dil dyes</td>
			<td>Molecular Probes</td>
			<td>Cat#C7000&#160;&#160;</td>
			<td>To label the bacteria</td>
		</tr>
		<tr>
			<td>Constant temperature shaking incubator</td>
			<td>Peiving Co., Ltd</td>
			<td>HZQ-X100</td>
			<td>Bacterial culture</td>
		</tr>
		<tr>
			<td>Database</td>
			<td>NCBI</td>
			<td>Bacteria and Archaea database</td>
			<td>Link: Archaea FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Archaea/ 
			Bacteria FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Bacteria/</td>
		</tr>
		<tr>
			<td>Disposable Pasteur pipette</td>
			<td>biosharp</td>
			<td>bs-xh-03l</td>
			<td>Used to change water, and transfer eggs</td>
		</tr>
		<tr>
			<td>Disposable petri dish</td>
			<td>biosharp</td>
			<td>BS-90-D</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>DNA kits</td>
			<td>Solaribio</td>
			<td>Cat#D1600</td>
			<td>Bacterial genomic DNA extraction kits&#160;</td>
		</tr>
		<tr>
			<td>Electric pipette</td>
			<td>SCILOGEX</td>
			<td>Levo me</td>
			<td>Change water</td>
		</tr>
		<tr>
			<td>Exiguobacterium</td>
			<td>NCBI database</td>
			<td>No.MK178504</td>
			<td>2019-JPP-ESN</td>
		</tr>
		<tr>
			<td>GZM</td>
			<td>Sea salt:LANDEBAO Co., Ltd.</td>
			<td>No Cat</td>
			<td>Composed of 1 L of water and 1.5 mL of sea salt solution (40 g/L), autoclaved. The content of sea salt in the GZM solution was 60 mg/L.</td>
		</tr>
		<tr>
			<td>Laboratory pure water system</td>
			<td>Hitech Co., Ltd</td>
			<td>Prima-S15</td>
			<td>Prepare the agents</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td>With fluorescent light to observe fish larvae</td>
		</tr>
		<tr>
			<td>PCR kits</td>
			<td>TIANGEN</td>
			<td>Cat#ET101</td>
			<td>Taq DNA Polymerase kit</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>LABSELECT&#160;</td>
			<td>sp-013-10</td>
			<td>Change water</td>
		</tr>
		<tr>
			<td>Povidone iodine (PVP-I)</td>
			<td>Aladdin</td>
			<td>Lot#H1217005</td>
			<td>Aqueous solution povidone iodine 0.4 g/L pure water.</td>
		</tr>
		<tr>
			<td>Timing converter</td>
			<td>PinYi Co., Ltd</td>
			<td>AL-06</td>
			<td>To regulate the light</td>
		</tr>
		<tr>
			<td>TSA plates</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp; 
			agar powder:BioFroxx.</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112; 
			agar powder:9002-18-0.</td>
			<td>TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, 6 g agar powder.</td>
		</tr>
		<tr>
			<td>TSB Aerobic</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp;</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112;</td>
			<td>TSB medium was prepared, wherein 400 mL medium included 6 g tryptone, 2 g soy peptone, and 2 g NaCl.</td>
		</tr>
		<tr>
			<td>TSB Anaerobic</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp;</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112;</td>
			<td>TSB medium was prepared and divided into the anaerobic tubes under the anaerobic system.</td>
		</tr>
		<tr>
			<td>Ultra-clean workbench</td>
			<td>Airtech</td>
			<td>SW-CJ-2FD</td>
			<td>Sterile treatment and testing</td>
		</tr>
		<tr>
			<td>Ultra-pure flow system for fish culture</td>
			<td>Marine Biological Equipment company</td>
			<td>No Cat</td>
			<td>Produce water for fish</td>
		</tr>
		<tr>
			<td>Vibrio</td>
			<td>NCBI database</td>
			<td>No.MK178501</td>
			<td>2019-JPP-ESN</td>
		</tr>
	</tbody>
</table>

generation, maintenance, and identification of germ-free zebrafish models from larvae to juvenile stages

ज़ेब्राफिश स्तनधारियों के साथ उनकी जीनोमिक समानताओं, अपेक्षाकृत स्वच्छ कोरियोन वातावरण में विकसित पारदर्शी भ्रूण, और कृंतक मॉडल की तुलना में लार्वा के बेहद तेजी से विकास के कारण विकास, प्रतिरक्षा और आंत माइक्रोबायोटा पर शोध के लिए मूल्यवान मॉडल के रूप में काम करते हैं। रोगाणु मुक्त (जीएफ) जेब्राफिश (डैनियो रेरियो) प्रदूषक विषाक्तता का मूल्यांकन करने और माइक्रोबियल कार्यों से संबंधित मानव जैसी बीमारी मॉडल स्थापित करने के लिए महत्वपूर्ण हैं। पारंपरिक रूप से उठाए गए (सीआर) मॉडल (आम पति में मछली) की तुलना में, जीएफ जेब्राफिश मेजबान माइक्रोबायोटा के अधिक सटीक हेरफेर की अनुमति देता है, सूक्ष्मजीवों और मेजबानों के बीच कारण संबंध निर्धारित करने में सहायता करता है। नतीजतन, वे इन रिश्तों की हमारी समझ को आगे बढ़ाने में महत्वपूर्ण भूमिका निभाते हैं। हालांकि, जीएफ जेब्राफिश मॉडल आमतौर पर प्रतिरक्षा समारोह और पोषक तत्वों के अवशोषण में सीमाओं के कारण प्रारंभिक जीवन चरणों (भ्रूण से लार्वा तक) के दौरान उत्पन्न और शोध किए जाते हैं। यह अध्ययन जीएफ भोजन (जैसे आर्टेमिया एसपी, ब्राइन झींगा) का उपयोग करके खिलाए बिना और लंबे समय तक खिलाने के साथ शुरुआती जीएफ जेब्राफिश मॉडल की पीढ़ी, रखरखाव और पहचान का अनुकूलन करता है। प्रक्रिया के दौरान, दैनिक नमूना और संस्कृति प्रदर्शन किया और प्लेटों और 16S rRNA अनुक्रमण सहित कई detections के माध्यम से की पहचान की गई. उत्पन्न मॉडल की गुणवत्ता और मात्रा सुनिश्चित करने के लिए जीएफ जेब्राफिश की सड़न रोकनेवाला दर, अस्तित्व और विकासात्मक सूचकांक दर्ज किए गए थे। महत्वपूर्ण बात, इस अध्ययन जीएफ मछली के लिए बैक्टीरियल अलगाव और संक्रमण तकनीक पर विवरण प्रदान करता है, GF खाद्य समर्थन के साथ किशोर चरणों के लिए लार्वा से GF मछली मॉडल के कुशल निर्माण को सक्षम. जैव चिकित्सा अनुसंधान में इन प्रक्रियाओं को लागू करके, वैज्ञानिक आंतों के जीवाणु कार्यों और मेजबान स्वास्थ्य के बीच संबंधों को बेहतर ढंग से समझ सकते हैं।

The microbiota (i.e., Archaea, Bacteria, Eukarya, and viruses) play crucial roles in maintaining host health and contributing to the development of various diseases by influencing physiological and pathological processes through symbiotic interactions within the intestinal barrier, epithelial surface, and mucin functions in individuals1,2,3. The composition of the microbiota across different life stages, from infancy to juvenility, adulthood, and aging, as well as its presence in various locations such as nares, oral, skin, and gut sites, is dynamically shaped by diverse habitats and environments4. The intestinal microbiota in organisms is involved in nutrient absorption, immune response, pathogen invasion, metabolic regulation, etc5,6. Studies on patients have demonstrated that disruptions in gut microbiota are related to human obesity, sleep disorders, depression, inflammatory bowel disease (IBD), neurodegenerative diseases (Parkinson&#39;s, Alzheimer&#39;s), aging, and various cancers7,8,9. Furthermore, interactive pathways between gut microbiota and hosts involve inflammatory factors, neurotransmitters, metabolites, intestinal barrier, and oxidative stress, as observed in previous research using mice and fish models10,11.
Recently, multiple bacteria-related approaches or therapies, including potential probiotics and fecal microbiota transplantation (FMT), have been explored for these disorders in clinical and animal models. These explorations are based on discoveries related to the microbiota-gut-brain/liver/kidney axis, microbiota-derived products, and altered receptor activity12,13. However, the development, various functions, and mechanisms of the microbiota-host system are still incompletely understood and identified due to the complexity of the microbial community and the challenge of generating powerful human-like disease models.
To address these issues, germ-free (GF) animal models were urgently proposed in the mid-19th century and primarily developed during the 20th century. Subsequent refinements, including antibiotic-treated and gnotobiotic models, along with advancements in microbial detection and observation technologies, further perfected these models14,15,16. GF animals, created by erasing their own background and avoiding environmental microbes, offer an excellent strategy for exploring the interactions between microorganisms and their hosts17. Through the application of animal models and refined protocols, researchers have successfully replicated similar microbial compositions found in patients in GF mice and fish. Additionally, other GF animal models, such as dogs, chickens, and pigs, provide diverse options as research subjects18,19,20,21. This approach has enabled investigations into the potential therapeutic effects of commensal microbiomes on various diseases, including cancer immunotherapy in humans16,18. GF models offer more accurate insights into the characteristics and mechanisms of specific bacterial colonization, migration, multiplication, and interaction within hosts. This provides crucial novel insights into the occurrence and development of microbiota-related diseases22,23. The history of establishing and applying GF zebrafish in microbial research has evolved from the reports of Rawls et al. in 2004 and Bates et al. in 2006 to Melancon et al.&#39;s protocol in 201716,24,25. However, the feasibility of adult or breeding GF models is still a prolonged process, accompanied by variable longevity, success rates, and health challenges.
Among various animal models, zebrafish (Danio rerio) stands out as a critical tool for both basic and biomedical research due to its advantageous similarity to human organs and genomics, short developmental cycle, high fecundity, and transparent embryos19,26. Zebrafish, serving as reliable human disease models, offer a visual representation of physiological and pathological processes in vivo, providing insights into the attractive features of host-microbe interactions. Notably, zebrafish exhibit distinct cell lineages, allowing imaging of intestinal physiology, microbial dynamics, gonads and reproductive development, maturation of the host immune system, behavior, and metabolism27. Zebrafish embryos develop within protective chorions until hatching, becoming larvae at 3 days post-fertilization (dpf). They actively hunt for food at 5 dpf and reach sexual maturity around 3 months post-fertilization (mpf)28. The first successful germ-free (GF) zebrafish, reported by Rawls et al.24, showed that larvae fed with autoclaved feed after yolk absorption exhibited tissue necrosis from 8 dpf and total death at 20 dpf. This indicated the effects of diet or the importance of considering exogenous nutrient supply in experiments involving long-term (&#62;7 dpf) GF fish29. Subsequent studies improved the generation protocol of GF fish, employing sterile food and methods perfected in different fish models16.
However, most research on GF zebrafish models has focused on early life stages, involving bacterial infection at 5 dpf for 24 h to 48 h, with samples collected before 7 dpf at the conclusion of the experiments25,30,31. It&#39;s widely acknowledged that the microbiota in organisms, including humans and zebrafish, is colonized at the beginning of life and shaped during growth and development. The composition remains stable at adult stages, with the roles of microbiota in the host being crucial throughout life, especially in aging, neurodegenerative, metabolic-related obesity, and intestinal disease aspects3. Thus, perspectives from GF animals with longer survival can provide insights into the mechanisms of microbial roles in host organ development and functions, considering the immature immune and reproductive systems of fish larvae in early life. While bacterial strains in zebrafish intestines have been isolated and identified in previous studies, offering the potential for infecting GF animal models to select probiotics or research bacterial functions in the host19,25, the generation and application of GF fish models have primarily been restricted to early life stages. This limitation, attributed to the complex production process, high maintenance costs, and associated issues with food and immunity, hinders research efforts aimed at investigating the developmental and chronic effects of microbiota in the host.
The survival rate, behavior, growth, maturation, and overall health of fish, especially in germ-free (GF) models, are significantly influenced by feeding practices, encompassing nutrition intake and absorption during the mouth-open period from early larvae to juveniles32,33. However, one of the challenges in GF fish husbandry is the scarcity of suitable sterile diets, limiting the effectiveness of nutritional support for sustaining the growth and survival of larvae. Resolving this issue is crucial to restoring the life of GF fish, considering their developmental defense mechanisms and weak digestion abilities due to the absence of an intestinal microbiome. In terms of food, live brine shrimp (Artemia sp.) emerges as the most suitable diet for mouth-open larvae to juvenile fish. It has been observed that fish fed with live brine shrimp exhibit higher growth and survival rates compared to those fed with cooked egg yolk or other natural and synthetic baits34. While early life models of GF fish can survive with yolk support and GF larvae models can be maintained with sterile feeding, generating long-term models from larvae to juveniles and reaching sexual maturity remains challenging. Additionally, flake or powder food is limited by unequal nutritional composition and can impact water quality. In contrast, live Artemia has advantages such as survival in both salt and freshwater, small size suitable for larvae to adults, ease of batching, and higher hatching quality35. Building upon previous methods16,24,30, we have simplified the complex treatment process and addressed the diet challenge by establishing easily incubated GF live Artemia sp. as sterile food for longer durations than early-life GF fish.
This study presents an optimized protocol covering (1) generation, (2) maintenance, (3) identification of sterile rate, and (4) maintenance and feeding to ensure the growth of germ-free (GF) zebrafish from embryos to larvae and juvenile stages. The results offer preliminary evidence on the hatching, survival, growth, and sterility of GF zebrafish, along with essential indices for GF Artemia sp. as sterile food. The detailed steps in model generation and preparation of sterile live foods provide crucial technical support for constructing and applying long-term GF fish models, as well as GF Artemia sp. in microbiota-host interaction research. The protocol addresses bacterial isolation, identification, and infection on GF fish models, outlining methods for bacterial fluorescence labeling and observing their colonization in fish intestines under a microscope. GF fish, gnotobiotic fish with bacterial infection, or transferred human microbiota models will undergo various detections to elucidate their functions and effects on host immunity, digestion, behavior, transcriptomic regulation, and metabolic aspects. In the long term, this protocol can be extended to different wild-type fish species, such as marine medaka, and potentially to other selected transgenic zebrafish lines correlated to specific tissues or diseases.

लेखक स्पॉटलाइट: माइक्रोबायोटा-होस्ट रिलेशनशिप स्टडीज के लिए रोगाणु मुक्त ज़ेब्राफिश की स्थापना

लार्वा से किशोर चरणों तक रोगाणु मुक्त जेब्राफिश मॉडल की पीढ़ी, रखरखाव और पहचान

The scope of this research is generation of novel, germ-free animal models, which will be helpful and powerful tools in investigation of various microbiome functions. In this field, the early germ-free fish models have been used recently, but the long-term models are still limited due to various culture conditions and the staff developmental defense. The current GF fish models are normally early life ones.Thus, the technologies including the sterile scale, the sample detection methods, and providing sterile food are very important challenges to overcome. The germ-free zebrafish model is crucial for studying host-microbiota interaction and their impacts on development and the immunity and disease. This model allows researchers to precisely manipulate the single gut bacteria and observe its effect on the transparent zebrafish embryos.It provides valuable insights into the roles of gut microbes inheriting the disease. In the future, our research will mainly focused on establishing a germ-free zebrafish model for entire life cycle to better understanding the function of the gut microbiome.

GF zebrafish मॉडल कुशलता से पिछले GF मछली मॉडल35 के आधार पर अनुकूलित प्रोटोकॉल के साथ, zebrafish के जोड़े द्वारा पैदा प्रचुर मात्रा में अंडे का उपयोग करके उत्पादित किया जा सकता है. एक एकल 6 अच्छी तरह से थाली पर्य...

Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

यह प्रोटोकॉल रोगाणु मुक्त (जीएफ) मछली भ्रूण प्राप्त करने और उन्हें किशोर चरण तक लार्वा से बनाए रखने के लिए प्राथमिक चरणों की रूपरेखा तैयार करता है, जिसमें नमूना लेना और उनकी बाँझ स्थिति का पता लगाना शामिल है। संक्रमण के साथ जीएफ मॉडल का उपयोग मेजबान स्वास्थ्य में रोगाणुओं की भूमिका को समझने के लिए महत्वपूर्ण है।

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

इस शोध का दायरा उपन्यास, रोगाणु मुक्त पशु मॉडल की पीढ़ी है, जो विभिन्न माइक्रोबायोम कार्यों की जांच में सहायक और शक्तिशाली उपकरण होंगे। इस क्षेत्र में, प्रारंभिक रोगाणु मुक्त मछली मॉडल का उपयोग हाल ही में किया गया है, लेकिन दीर्घकालिक मॉडल अभी भी विभिन्न संस्कृति स्थितियों और कर्मचारियों के विकास की रक्षा के कारण सीमित हैं। वर्तमान GF मछली मॉडल सामान्य रूप से प्रारंभिक जीवन वाले हैं।इस प्रकार, बाँझ पैमाने, नमूना का पता लगाने के तरीके, और बाँझ भोजन प्रदान करने सहित प्रौद्योगिकियां दूर करने के लिए बहुत महत्वपूर्ण चुनौतियां हैं। रोगाणु मुक्त जेब्राफिश मॉडल मेजबान-माइक्रोबायोटा इंटरैक्शन और विकास और प्रतिरक्षा और बीमारी पर उनके प्रभावों का अध्ययन करने के लिए महत्वपूर्ण है। यह मॉडल शोधकर्ताओं को एकल आंत बैक्टीरिया में सटीक हेरफेर करने और पारदर्शी जेब्राफिश भ्रूण पर इसके प्रभाव का निरीक्षण करने की अनुमति देता है।यह बीमारी को विरासत में लेने वाले आंत रोगाणुओं की भूमिकाओं में मूल्यवान अंतर्दृष्टि प्रदान करता है। भविष्य में, हमारा शोध मुख्य रूप से आंत माइक्रोबायोम के कार्य को बेहतर ढंग से समझने के लिए पूरे जीवन चक्र के लिए एक रोगाणु मुक्त जेब्राफिश मॉडल स्थापित करने पर केंद्रित होगा।

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Research

JoVE Journal

Developmental Biology

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Generation of Germ-Free Zebrafish Models from Embryos

भ्रूण से रोगाणु मुक्त जेब्राफिश मॉडल की पीढ़ी

GF Artemia nauplii Preparation for Chronic GF Zebrafish Nutrition

To begin, obtain 0.25 grams of Artemia cysts and rinse them with 2.4 grams per liter sodium hypochlorite for five minutes. Filter the rinsed cysts with a sterilized ultra dense filter and wash with sterilized ultrapure water for one to two minutes. After three washes, transfer them to 100 milliliters of 2.5%sterile saline and mix well.Pack 50 milliliters of treated Artemia cyst mix into a sterile 100-milliliter bottle. Using a film, seal the bottle thoroughly and place it in a shaking incubator with light. Next, using a Pasteur pipette, extract 30 milliliters of hatching Artemia nauplii from the middle and lower layers.After filtering, wash the Artemia three times with sterile ultrapure water for about one minute, and add four milliliters of sterile ultrapure water. Finally, using a disposable Pasteur pipette, retrieve actively swimming Artemia from the upper layer, wash, and prepare the solution for feeding. Microscopic observation confirmed efficient hatching of Artemia cysts after the treatment.