A Novel, Reliable Method for Repeated Blood Collection from Aquarium Fish

Zang, Liqing; Shimada, Yasuhito; Nishimura, Yuhei; Tanaka, Toshio; Nishimura, Norihiro

doi:10.1089/zeb.2012.0862

Cited by 69 publications

(51 citation statements)

References 37 publications

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“…Fasting blood glucose increased with overfeeding in both groups, consistent with the data from Zang et al (41). However, by the 2nd wk of overfeeding the zMIR fish had significantly elevated blood glucose compared with the controls (P Ͻ 0.01, t-test; Fig.…”

Section: Generation Of a Skeletal Muscle Insulin-resistant Zebrafish supporting

confidence: 90%

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Maddison

Joest

Kammeyer

et al. 2015

American Journal of Physiology-Endocrinology and Metabolism

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Section: Generation Of a Skeletal Muscle Insulin-resistant Zebrafish supporting

confidence: 90%

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Maddison

Joest

Kammeyer

et al. 2015

American Journal of Physiology-Endocrinology and Metabolism

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“…Similar to mammals, zebrafish also developed abnormal glycolipid metabolism when fed a high-fat diet. The changes in fasting blood glucose of each individual showed individual differences in the response to overfeeding similar to that in humans 15,27 . This result indicates that an investigation of the temporal changes in blood biochemical parameters of individual fish will provide a good opportunity to determine the individual differences in metabolic disorders such as obesity and type 2 diabetes mellitus, thus further confirming its value as an animal model for human disease.…”

Section: Discussionmentioning

confidence: 59%

“…We investigated these effects using measurements of blood hemoglobin level (Figure 4). As we have shown in a previous publication 15 , adult male fish weighing approximately 0.5 g each were assigned to four groups. Repeated blood sampling (2 µl each time) of the same individual fish once daily for 7 days (for a total of seven blood samples) resulted in significant decrease (p <0.01) in hemoglobin levels from 10.82 ± 0.78 g/dl to 2.38 ± 0.8 g/dl.…”

Section: Representative Resultsmentioning

confidence: 99%

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Repeated Blood Collection for Blood Tests in Adult Zebrafish

Zang

Shimada

Nishimura

et al. 2015

JoVE

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Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel "repeated" blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be ≤0.4% of body weight every week or ≤1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism. Video LinkThe video component of this article can be found at

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“…For blood glucose in adult animals, fish were either fasted overnight or fed as normal and blood was collected 30 min after the meal. Blood was collected from the dorsal aorta along the trunk of the fish as indicated in Zang et al (2013). Blood glucose was determined as described (Li et al 2014).…”

Section: Feeding and Glucose Measurementmentioning

confidence: 99%

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

et al. 2015

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Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.KEYWORDS CRISPR/Cas9; conditional mutagenesis; glucose homeostasis; retinal regeneration; zebrafish C ONDITIONAL gene inactivation is necessary for determining physiological functions of genes whose conventional mutation causes embryonic lethality or multiorgan defects. It has been widely used in mice because of the availability of embryonic stem (ES) cells and large collections of both ES cell lines and animals that carry conditional alleles. The conditional alleles usually contain strategically placed Cre or Flp target sites in introns that permit deletion of the intervening exon(s) (Gu et al. 1994). Some conditional alleles harbor a Cre and/or Flp invertible gene trap in an intron that allows an on/off switch of transcription (Schnutgen et al. 2005;Ni et al. 2012). The function or expression of these alleles is "switched" off in the presence of Cre or Flp activity. In Drosophila, conditional gene inactivation can be achieved using RNA interference (RNAi), and genome-wide libraries of RNAi transgenes are available (Dietzl et al. 2007;Ni et al. 2008). For the increasingly popular zebrafish model, however, only limited conditional alleles generated from invertible gene trapping are available (Ni et al. 2012). The lack of ES cells and the inefficiency of RNAi in zebrafish necessitate new approaches for targeted conditional gene inactivation.CRISPR/Cas9 mutagenesis has been applied to many model systems from cultured cells to whole organisms (Doudna and Charpentier 2014;Hsu et al. 2014). The system only requires a two-component RNA-protein complex (RNP): a single guide RNA (sgRNA) that identifies the target through base pairing and the Cas9 endonuclease that generates double-strand breaks (DSBs) at the target site upon sgRNA-target base pairing. CRI...

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A Novel, Reliable Method for Repeated Blood Collection from Aquarium Fish

Cited by 69 publications

References 37 publications

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

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