WO2020218731A1 - Modèle animal transgénique à invalidation génétique fam19a5l et son procédé de production - Google Patents

Modèle animal transgénique à invalidation génétique fam19a5l et son procédé de production Download PDF

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WO2020218731A1
WO2020218731A1 PCT/KR2020/002957 KR2020002957W WO2020218731A1 WO 2020218731 A1 WO2020218731 A1 WO 2020218731A1 KR 2020002957 W KR2020002957 W KR 2020002957W WO 2020218731 A1 WO2020218731 A1 WO 2020218731A1
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fam19a5l
zebrafish
animal model
gene
transgenic animal
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박해철
정인영
성재영
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고려대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to a Fam19a5l gene knock-out transgenic animal model and a manufacturing method thereof.
  • Neuropeptide is a peptide with a short amino acid sequence that is mainly produced and secreted by neurons, and transmits signals into cells through binding to a specific G protein-coupled receptor (GPCR). Through the signal transmission between these nerve cells, they perform physiological regulation functions such as fear/anxiety, reward, feeding, and sleep in the brain. Thus, abnormalities in neuropeptide action can cause various diseases in the nervous system. Until now, many neuropeptides have been discovered and studied, but not much is known about how they function and regulate the physiological functions of the brain. Therefore, discovery of new neuropeptides and research on physiological activity functions are essential for the identification of physiological regulatory functions in the brain.
  • zebrafish (Danio rerio) is a three-year-old tropical freshwater fish, has 25 pairs of chromosomes, and has genes preserved even though it was separated from a common ancestor of civilization 300 million years ago in evolution, it possesses almost all genes that humans have have. Due to these characteristics, zebrafish began to be used as a developmental biology model in the 1980s.
  • Zebrafish are less expensive than other animal models, and as vertebrates, they are genetically very close to humans compared to fruit flies or linear animals.
  • a large number of fertilized eggs are produced, enabling highly efficient research, and the embryo is transparent and has a short development period of 48 hours, allowing real-time observation.
  • organ-specific expression of a biomarker such as a fluorescent protein
  • the neuropeptide FAM19A5 homologous genes were identified using a zebrafish animal model, and the Fam19a5l gene expression neural circuit, one of the homologous genes of FAM19A5, was analyzed and Fam19a5l function was studied. Through this, we tried to investigate the function of the neuropeptide Fam19a5l gene to regulate pain response.
  • pain is a sensation caused by harmful stimuli, and although there have been many studies on nociceptive reactions in mammals, the development of new painkiller drugs is not done much, and because it takes a lot of cost and time to reduce it recently. Attempts are ongoing, and zebrafish are attracting attention as an alternative.
  • zebrafish as in mammals, various pain models have been established, and pain research is being actively conducted. The physiology of pain in zebrafish is well established, and it is known that it is almost similar to that of mammals.
  • the zebrafish pain model can be established through drug, temperature, and electrical stimulation, and a pain model using drugs or temperature is currently established.
  • Drugs can cause pain in zebrafish through acetic acid, mustard oil, cinnamaldehyde, and histaminee, which are well known in mammals, and are treated with analgesics such as morphine. It has been found that the eye pain response decreases. There is also a model that measures the pain response by measuring the degree of avoidance against low and high temperatures. This pain model is called larva. It has the advantage of being able to perform both in and in the adult. However, these pain models have been established relatively recently, and little research on the level of new genes involved in pain response has been made.
  • FAM19A (Family with sequence similarity 19 (chemokine (CC motif)-like), member A) family genes consist of a total of five paralogous genes from FAM19A1 to FAM19A5, and the conserved cysteine of chemokine. It encodes a secretory protein with a cysteine residue.
  • S1PR2 sphingosine-1-phosphate receptor 2
  • the present inventors completed the present invention by confirming the expression pattern of the neuron-related gene in the zebrafish knocking out the Fam19a5l gene, one of the homologous genes of FAM19A5, and observing the relationship with the pain response.
  • An object of the present invention is to provide a transgenic animal model in which the Fam19a5l gene is knocked out.
  • Another object of the present invention is 1) preparing a Fam19a5l gene knock-out mixture; 2) introducing the mixture of step 1) into the fertilized egg; And 3) selecting a Fam19a5l gene knockout embryo from the fertilized egg of step 2).
  • transgenic animal model in which the Fam19a5l gene is knocked out.
  • the animal may be one or more selected from the group consisting of zebrafish, mouse, rat, pig, and monkey.
  • the animal may be a zebrafish.
  • trpa1a and trpa1b gene expression may be reduced.
  • ngfa nerve growth factor a gene expression may be reduced.
  • the animal model may have a reduced pain response.
  • the pain response is induced by one or more methods selected from the group consisting of acetic acid, mustard oil, cinnamaldehyde, histamine drug, temperature, and electrical stimulation. It may be a transgenic animal model.
  • a method of producing a zebrafish animal model for pain reduction is provided.
  • the mixture of step 1) may be a method for producing a zebrafish animal model, which is prepared by combining a guide RNA and a Cas9 protein.
  • the Fam19a5l gene knockout embryo prepared in step 3 after raising the Fam19a5l gene knockout embryo prepared in step 3) into an adult, it may be a method of producing a zebrafish animal model, further comprising the step of preparing a homozygous transformant by crossing it with a wild type. .
  • the Fam19a5l gene knockout zebrafish animal model can be prepared to discover the function of the Fam19a5l gene involved in the pain response.
  • Fam19a5a, Fam19a5b, and Fam19a5l three kinds of zebrafish homologous genes of human FAM19A5 exist.
  • Figure 2 shows the expression pattern of the zebrafish homologous gene of FAM19A5, (B-H') shows that Fam19a5l is expressed in a specific region of the zebrafish nervous system, which will play a specific physiological role in the nervous system. This is a picture showing the possibility.
  • 3A shows the location of the Fam19a5l protein in vivo in the zebrafish nervous system. It is a schematic diagram of a strategy for tracking, and (B-C''') is a photograph showing the location of the Fam19a5l protein in vivo in the zebrafish nervous system.
  • Figure 4 is a schematic diagram showing the manufacturing process of Fam19a5l:egfp-CAAX BAC DNA.
  • Figure 5 is a cross-sectional view showing a Fam19a5l-specific green fluorescent protein-expressing transgenic zebrafish was prepared.
  • FIG. 6 is a photograph showing the characteristics of cells expressing the Fam19a5l protein in the zebrafish brain.
  • FIG. 7 is a photograph showing the expression of the Fam19a5l protein in zebrafish sensory nerve cells and dorsal intermediate nerve cells.
  • FIG. 8 is a diagram showing a predicted peptide sequence made from the Fam19a5l gene knockout zebrafish in which 20 bases are lost from the nucleotide sequence represented by SEQ ID NO: 7 of Exon1.
  • 9A and 9B It is a graph showing the results of an experiment for controlling nociception by comparing wild-type zebrafish individuals with Fam19a5l knockout fry.
  • FIG. 10 is a graph showing the results of confirming the expression pattern of neuronal cell-related genes through quantitative reverse transcription polymerase chain reaction (RT-PCR) in Fam19a5l gene knockout cheer.
  • RT-PCR quantitative reverse transcription polymerase chain reaction
  • the term “knock-out” refers to preventing the expression of a specific gene, and means preventing the specific gene from functioning by causing a specific gene to be deleted or replaced with a mutated gene.
  • the present invention provides a transgenic animal model in which the Fam19a5l gene is knocked out.
  • the transgenic animal model of the above manufacturing method is preferably a zebrafish, but is not limited thereto, and all animals including mice, rats, pigs, monkeys, horses, cows, sheep, dogs, cats, rabbits, etc. may correspond to this. have.
  • the present invention provides a zebrafish transgenic animal model in which the Fam19a5l gene is knocked out.
  • the transgenic animal model in which the Fam19a5l gene of the present invention is knocked out may be characterized by reduced trpa1a and trpa1b gene expression.
  • ngfa nerve growth factor a
  • trpa1a and trpa1b mRNA expression were reduced by 30-50% in Fam19a5l knockout fry compared to zebrafish wild-type fry.
  • tachykinin 1 tac1
  • TRPA1 trpa1a and trpa1b
  • NGF nerve growth factor
  • Fam19a5l gene knockout transgenic animal model representing the expression pattern of the neuron-related gene may be characterized by a reduced pain response.
  • the pain response may be induced by one or more methods selected from the group consisting of acetic acid, mustard oil, cinnamaldehyde, histamine drug, temperature, and electrical stimulation. Not limited.
  • step 2) introducing the mixture of step 1) into the fertilized egg
  • the knock-out mixture of step 1) preferably includes a genetic scissors prepared by combining guide RNA and Cas9 protein, and the genetic scissors are ZFN (zinc-finger nuclease), TALEN It is preferable to include any one selected from the group consisting of (transcription activator-like effector nuclease) and CRISPR/Cas9 (clustered regulary interspaced short palindromic repeats/CRISPR-associated protein-9). According to an embodiment of the present invention, CRISPR/Cas9 was selected and used.
  • the mutagenesis carrier comprising CRISPR/Cas9 preferably knocks out the Fam19a5l gene, but includes all cases in which not only the entire Fam19a5l sequence but also part of the sequence is knocked out.
  • the Fam19a5l gene knockout mixture was used by microinjection, electroporation, particle bombardment, and sperm-mediated gene transfer. , Viral infection, direct muscle injection, insulator, and transposon can be introduced into a fertilized egg.
  • a vector containing the nucleotide sequence knocked out of the Fam19a5l gene was injected into a zebrafish fertilized egg using a microinjection method, and a transformed zebrafish animal model was prepared.
  • the step of selecting the Fam19a5l gene knockout embryo of step 3) may be selected according to various methods well known in the art. Examples of such a selection method include a PCR method using tail genomic DNA as a template or a method of visually discriminating abnormal symptoms of a knockout embryo, and it is preferable to visually determine the abnormal symptoms.
  • the present invention may further include the step of preparing a homozygous transformant by raising the Fam19a5l gene knockout embryo prepared in step 3) into an adult, and then crossing it with a wild type.
  • Example 4 of the present invention an animal model of the fam19a5l gene knockout transgenic zebrafish was prepared, and the behavior of the Fam19a5l gene knockout zebrafish was observed in Experimental Example 1, and a zebrafish trait showing a characteristic of reduced pain response The converted animal model could be confirmed.
  • Example 1 Construct a pain model
  • Wild-type zebrafish, Tg(Fam19a5l:egfp-caax), Tg(huc:gal4), and Tg(uas:egfp) transgenic zebrafish were used in this study.
  • Adult and embryonic zebrafish were bred in a breeding room with 14 hours light and 10 hours dark circumference at 28.5 degrees Celsius.
  • zebrafish embryos and larva were defined and used according to the morphological characteristics of the time after fertilization (hours post-fertilization, hpf) or days post-fertilization (dpf). Embryo media was treated to prevent formation of 0.003% 1-phenyl-2-thiourea (PTU) pigment in embryos 24 hours after fertilization.
  • PTU 1-phenyl-2-thiourea
  • the amino acid cleavage site known through previous studies was marked with *, and it was confirmed that the zebrafish FAM19A5 homologous genes also preserved this cleavage site. It was confirmed that most amino acid sequences of the mature peptide of the zebrafish FAM19A5 gene were similar to those of humans and rodents.
  • RT-PCR reverse transcription polymerase chain reaction
  • the Fam19a5a, Fam19a5b, and Fam19a5l gene primers used in reverse transcription polymerase chain reaction (RT-PCR) are shown in Table 1.
  • each gene primer was prepared using the base sequence obtained from Ensemble (Fam19a5a: ENSDARG00000069160, Fam19a5b: ENSDARG00000045608, Fam19a5l: ENSDARG00000068100).
  • Fam19a5a ENSDARG00000069160
  • Fam19a5b ENSDARG00000045608
  • Fam19a5l ENSDARG00000068100.
  • DNA amplified through polymerase chain reaction from cDNA of fry 5 days after fertilization was cloned into pGEM®-T Easy Vector System (Promega).
  • pGEM®-T Easy Vector System Promega
  • the temporal and spatial expression patterns of the Fam19a5l gene were confirmed by performing a whole-mount in situ hybridization.
  • RNA probes specific for Fam19a5l RNA were prepared.
  • the vector in which the Fam19a5l gene was cloned was cut with restriction enzyme SacII (New England Biolabs) and linearized, and then, using a mixture labeled with SP6 RNA polymerase and digoxygenin (Dig RNA Labeling Mix, Roche). The probe was synthesized according to the manufacturer's protocol.
  • PBStx containing 10 to 50 ⁇ g/ml proteinase K was treated for 5 to 30 minutes, and then fixed at room temperature for 20 minutes or more with 4% paraformaldehyde/PBS. After washing 4 times with PBStx for 10 minutes each, 500 ⁇ l hybridization solution heated at 60 to 65° C., 50% formamide, 5 X SSC, 5 mg/ml torula RNA, 50 ⁇ g/ Ml heparin, citric acid, pH6, 0.1%, 0.2% Triton-X) was added and washed at 60°C for 5 minutes.
  • hybrid solution 500 ⁇ l was replaced again and prehybridized at 60 to 65°C for 2 to 3 hours or more, and then 200 to 300 ng of a probe was added to hybridize at 60 to 65°C for 18 hours or more ( hybridization).
  • the probe was removed at 60 to 65° C., and 100%, 75%, 50% and 25% mixed solutions/2X SSCtw (2xSSC buffer + 0.1% tween-20) were sequentially washed every 10 minutes. At the same temperature, it was replaced with 2 X SSCtw and washed twice for 15 minutes, followed by washing with 0.2 X SSCtw 4 times every 20 to 30 minutes and 0.1 X SSCtw for 15 minutes.
  • the fertilized eggs after staining were washed 4 times with PBStw for 10 minutes each, and then fixed in 4% formaldehyde at 4° C. for 14 to 18 hours. Photographing of stained zebrafish embryos and fry was performed after washing four times with PBStw for 10 minutes each, and then gradually changing the solution with 25%, 50%, and 75% glycerol/PBStw.
  • Fam19a5l is expressed in a specific area of the nervous system and predicted the possibility of performing a specific physiological role in the nervous system (see Figs. 2B-H').
  • Fam19a5l in the second day after fertilization of an adult zebrafish is found in the brain in the lower cortex (subpallium), cortex (pallium), dorsal thalamus, cerebellar plate, optical tectum, and medulla. oblongata) region (Fig. 2B-C', black arrow), as well as the spinal cord (Fig. 2D, red arrow).
  • Fam19a5l at the 3 day stage after fertilization was expressed at the 2 day stage including the preoptic area, midbrain tegmentum, and hypothalamus (Fig. 2E-F ⁇ , black arrows). Similar to but observed in a wider area, it was confirmed that it was expressed in the ventricular zone (Fig. 2F ⁇ , black arrow head) and the retina of the eye (Fig. 2F, black arrow). ).
  • the human FAM19A5 protein is a secreted peptide, has a signal peptide consisting of 43 amino acids, and that the mature peptide starts with the 44th threonine residue.
  • the signal peptide cleavage site of FAM19A5 in humans and rodents is evolutionarily conserved in the zebrafish homologous gene (see FIG. 1).
  • Tg(huc:gal4); Tg(uas:egfp)) that expresses the Gal4 protein specifically for neurons and expresses the green fluorescent protein through the UAS sequence by using 5xuas:Fam19a5l:mcherry plasmid DNA. It was microinjected into the cell-phase fertilized egg. Thereafter, the Fam19a5l-red fluorescent binding protein was tracked in cells or tissues through in vivo imaging at 3 days after fertilization (see FIG. 3).
  • Fam19a5l-red fluorescent binding protein As a result, only a part of the Fam19a5l-red fluorescent binding protein was observed when it was present inside the cell body of HuC+/DAPI+ neurons (Fig. 3B' white arrowhead), and most of the Fam19a5l-red fluorescent binding protein was HuC+. /DAPI + was present in the vicinity or adjacent to the neuron (Fig. 3B', B'', C''', white arrows). In addition, this binding protein was observed in a place where there were no neurons around and no cell nucleus was also present (GFP-/DAPI) (Fig. 3B'-B ⁇ '.C'', yellow arrow). These results showed that many Fam19a5l-red fluorescent binding proteins were found more outside the cell than inside. Therefore, it was confirmed that zebrafish Fam19a5l is also a secretory peptide.
  • Example 3 Preparation of transgenic zebrafish expressing Fam19a5l gene-specific fluorescent protein
  • a transformed zebrafish (Fam19a5l:egfp-caax)) that expresses the Fam19a5l gene-specific fluorescent protein
  • BAC bacterial artificial chromosome
  • Fam19a5l BAC DNA Fam19a5l:egfp-caax BAC DNA was prepared using an E. coli-based homologous recombination system. This modified BAC DNA was made by binding the DNA sequence encoding the membrane EGFP (EGFP-CAAX) to the start codon ATG site of the open reading frame (ORF) of the Fam19a5l gene of BAC DNA.
  • Fam19a5l:egfp-caax BAC DNA preparation process is shown in FIG.
  • the target plasmid DNA was cloned through a gateway system.
  • the target plasmid DNA was subjected to a multi-site gateway LR reaction according to the manufacturer's protocol.
  • each entry vector was cloned through the BP reaction.
  • Each 5'- and 3'-entry vector was prepared by inserting an upper or lower 500 base-pair from the initiation codon to serve as a homology arm.
  • the intermediate entry vector was prepared by inserting egfp-caax encoding the membrane green fluorescent protein and the nucleotide sequence encoding the antibiotic kanamycin resistance gene used as a colony selection marker.
  • Fam19a5l:egfp-caax BAC DNA was prepared by removing the kanamycin resistance gene through a flip enzyme recombination (flipase(flp) recombination) process.
  • the Fam19a5l:egfp-caax BAC DNA prepared in Example 3-1 was microinjected into a zebrafish 1-cell stage fertilized egg to prepare a transformed zebrafish (Tg (Fam19a5l:egfp-caax)) (see FIG. 5). ). After raising the zebrafish fry to an adult (Founder, F0), they were bred with wild-type zebrafish to obtain zebrafish embryos. A zebrafish (F1) showing a Fam19a5l-specific green fluorescent protein was identified, and stable transgenic zebrafish were prepared through breeding and crossing of the zebrafish (see FIG. 5).
  • Fig. 5 shows a lateral view (A) and a dorsal view (A') of a Tg (Fam19a5l:egfp-caax) fry at the second day after fertilization.
  • Zebrafish embryos and fry are anesthetized with an anesthetic (ethyl 3-aminobenzoate methanesulfonate salt, 200 mg/L (MS222, Sigma)), and then put in 4% paraformaldehyde/PBS (1X phosphate buffered saline) 4 It was fixed for 12 to 18 hours or longer at °C. Fixed embryos and fry are made into blocks by immersing them in 1.5% agarose/5% sucrose solution, and then 12 to 18 hours at 4°C until the blocks are equilibrated and settled in 30% sucrose solution. It was stored longer.
  • anesthetic ethyl 3-aminobenzoate methanesulfonate salt, 200 mg/L (MS222, Sigma)
  • the secondary antibody was diluted in a blocking solution and reacted at 4° C. for 12 to 18 hours or longer. After the reaction, in order to stain the nuclei of tissue cells, they were reacted at room temperature for 15 minutes with DAPI diluted in a blocking solution. Then, after washing with PBS for 1 to 2 hours, the slide glass was covered with a cover slide on which 150-200 ⁇ l of a 75% glycerol/25% PBS solution was placed, and fluorescence photographs were taken.
  • the primary and secondary antibodies used in the experiment are shown in Table 2 below.
  • DAPI D1306, Invitrogen
  • the shape of the cells was similar to the shape of a radial glial cell existing in the ventricular region, and it was confirmed that the cells express BLBP, which is a marker for radioglia (see FIGS. 6C to E). Therefore, it was confirmed that Fam19a5l was expressed in some radioglial cells of the initiation cover and thalamus. It was confirmed that Fam19a5l was expressed in the initiation cover of radioglia and in some regions of the thalamus, and was expressed in both neuron and radioglia.
  • Fam19a5l is the lateral pallium and medial pallium of the forebrain, the preoptic area, the thalamus, and the optical tectum of the midbrain. , It was confirmed that it is expressed in the regions of the middle and marginal vagal neurons of the hindbrain. This showed the same results as the Fam19a5l expression pattern, which was mostly confirmed by whole-tissue RNA in-place hybridization.
  • Fam19a5l from zebrafish embryo to cheering stage was expressed in some neurons of trigeminal ganglion mediating chemosensation (FIG. 7A, B white arrow), and equilibrium -It was confirmed that it is expressed in part of the statocoustic ganglion neuron (Fig. 7A, yellow arrow).
  • the expression of Fam19a5l was also confirmed in the retinal ganglion cells of the eye in the cheering stage (Fig. 7B, blue arrow).
  • Fam19a5l is also expressed in a part of the dorsal root ganglion neuron that senses mechanical stimulation or nociception (Fig. 7C, white arrow), and is known to be involved in sensory information processing in the spinal cord. It was confirmed that it was also expressed in part of the cells (dorsal interneuron) (Fig. 7C, yellow arrow).
  • Fam19a5l + part of the nerve cells and most of the peripheral nervous system Fam19a5l + dorsal root ganglion neurons were HuC/D + /Isl1/2 + /Pax2 - neurons (Fig. 7E, F, arrows).
  • isl2b is known to be expressed in dorsal longitudinal interneuron and dorsal root ganglion neuron. Therefore, it was confirmed that Fam19a5l + dorsal intermediate nerve cells in the spinal cord are dorsal longitudinal interneurons, and Fam19a5l + /Isl1/2 + nerve cells in the peripheral nervous system are dorsal root ganglion neurons. I did.
  • Fam19a5l was expressed in various sensory nerve cells and intermediate nerve cells that process sensory nerves in the zebrafish nervous system. Therefore, Fam19a5l predicted the possibility that it could play a role in sensory and nociceptive responses.
  • Fam19a5l gene knock-out zebrafish was prepared using the CPISPR/Cas9 gene scissors technology.
  • the base sequence represented by SEQ ID NO: 7 present in exon 1 near the start codon in the Fam19a5l gene was selected as a target sequence, and guide RNA (guide RNA) was prepared (see Fig. 8).
  • guide RNA guide RNA
  • a primer represented by SEQ ID NO: 7 in Table 3 was used.
  • Fam19a5l exon1 F 5'-GGGATGCGGGCGCTCTGTGCCGG-3'
  • a primer represented by SEQ ID NO: 8 containing a short genomic DNA nucleotide sequence including both sides of the Cas9 target site was prepared and then amplified by polymerase chain reaction. Then, the amplified sample was raised to a high temperature (95° C.) and then slowly dropped, followed by a heteroduplex experiment, and treated with T7 internal nucleotide cleavage enzyme I. Thereafter, F0 candidate zebrafish having mutations in which the size of the amplified DNA band with the original size and the two types of DNA bands were cut were selected when observed through 2% gel electrophoresis experiment.
  • the extracted genomic DNA was amplified by polymerase chain reaction (PCR) using a primer represented by SEQ ID NO: 8, and then cloned into pGEM®-T Easy Vector (Promega). .
  • PCR polymerase chain reaction
  • SEQ ID NO: 8 a primer represented by SEQ ID NO: 8
  • pGEM®-T Easy Vector Promega
  • the sequence in which the mutation occurred was analyzed through DNA sequencing of the cloned vector.
  • the primers used in the pGEM®-T Easy Vector (Promega) cloning and T7 endonuclease I assay are shown in Table 4 below.
  • Fam19a5l gene was knocked out in the prepared zebrafish (see FIG. 8).
  • a knockout zebrafish in which 20 bases represented by SEQ ID NO: 7 present in exon 1 of the Fam19a5l gene were deleted (20 bp deletion) was identified.
  • a premature stop codon was generated in the Fam19a5l gene, resulting in abnormal short peptides or immature stop codon-dependent mRNA degradation (nonsense mRNA decay).
  • the Fam19a5l gene knockout zebrafish prepared in Example 4 was observed to observe the behavior of the Fam19a5l gene loss to determine the effect of the zebrafish behavior.
  • Zebrafish cheer behavior analysis was performed using Danio Vision (Noldus), which includes an automatic behavior tracking analysis program Ethovision XT12 (Noldus).
  • Danio Vision (Noldus)
  • Ethovision XT12 (Noldus)
  • genotype analysis of all fry was performed after the behavioral experiment was completed.
  • wild-type offspring and Fam19a5l knockout fry were subjected to a nociceptive reaction test at the 3 day stage after fertilization, and the fry motility test was performed at the 5 day stage after fertilization.
  • the fry were raised in a breeding room with a 14 hour light/10 hour dark circumference at 28.5°C, and transferred to a 48 well-plate one day before the experiment.
  • Fam19a5l gene knockout zebrafish prepared in Example 4 it was attempted to confirm the behavioral change for the nociceptive reaction.
  • a method to check the nociceptive reaction a method of measuring the increase in motility caused by stimulation by treating a chemical that gives harmful stimulation in the early stage of zebrafish fry without motility was used.
  • Mustard seed oil (mustard oil, allyl isothiocyanate (AITC), 377430, Sigma-Aldrich) was used as a noxious drug to induce a locomotor response. It is known to act as an agonist of the TRPA1 channel protein. After fertilization, 100 ⁇ m of mustard seed oil was treated with wild-type progeny and Fam19a5l knockout fry on the third day after fertilization, and changes in motility were observed (see FIGS. 9A and 9B).
  • the control group was treated with 1% DMSO (Dimethyl sulfoxide, Biosesang, D1022), and the experimental group was treated with 100 ⁇ m mustard seed oil (100 ⁇ m allyl isothiocyanate in 1% DMSO).
  • DMSO Dimethyl sulfoxide
  • the experimental group was treated with 100 ⁇ m mustard seed oil (100 ⁇ m allyl isothiocyanate in 1% DMSO).
  • the fry behavior before the drug treatment was recorded for 5 minutes in the light condition, and the fry behavior after the drug treatment was also recorded for 5 minutes in the light condition.
  • the Fam19a5l gene knockout zebrafish prepared in Example 4 was used to confirm how Fam19a5l regulates the nociceptive response.
  • Mustard seed oil acts as an agonist of the TRPA1 channel, and it is known that the TRPA1 homology gene in zebrafish is expressed in trigeminal ganglion neurons. Therefore, it was hypothesized that Fam19a5l could regulate the expression of trpa1, and this was to be verified.
  • TRPA1 is increased in expression by nerve growth factor (NGF) in trigeminal ganglion neurons. Therefore, we tried to confirm whether the decrease in mRNA expression of trpa1a and trpa1b in Fam19a5l knockout zebrafish was due to the decrease in the expression of nerve growth factor.
  • NGF nerve growth factor
  • the quantitative reverse transcription polymerase chain reaction experiment was performed using a LightCycler 96 instrument (Roche).
  • the cDNA used in the experiment was synthesized from RNA extracted from wild-type progeny and Fam19a5l knockout fry at the 3 day stage after fertilization in the same manner as in the behavioral experiment.
  • Each reaction mixture including 2.5 ⁇ l of template cDNA, 0.2 ⁇ m primer, and 2X Fast Start Essential DNA Green Master Mix (Roche) is 40 cycles of 95 °C 10 minutes, 95 °C 10 seconds, 60 °C 10 seconds, 72 °C 10 seconds It was amplified through a program step consisting of.

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un modèle animal transgénique à invalidation génétique Fam19a5l et son procédé de production et, plus spécifiquement, concerne un poisson zèbre transgénique dans lequel le gène Fam19a5l a été invalidé, et un procédé de production du poisson zèbre.
PCT/KR2020/002957 2019-04-23 2020-03-02 Modèle animal transgénique à invalidation génétique fam19a5l et son procédé de production WO2020218731A1 (fr)

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KR10-2019-0047300 2019-04-23
KR1020190047300A KR102328773B1 (ko) 2019-04-23 2019-04-23 Fam19a5l 유전자를 녹아웃 시킨 제브라피쉬 동물모델 및 이의 제조방법

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Citations (4)

* Cited by examiner, † Cited by third party
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KR20130094255A (ko) * 2012-02-15 2013-08-23 고려대학교 산학협력단 신경교세포 생성 조절에 관여하는 fam19a5의 약제학적 용도
KR20160101786A (ko) * 2015-02-17 2016-08-26 울산대학교 산학협력단 Ｆａｍ１９ａ５ 단백질을 포함하는 비만 예방 또는 치료용 조성물 및 이를 이용한 비만 치료제의 스크리닝 방법
KR20160143913A (ko) * 2015-06-04 2016-12-15 충남대학교산학협력단 Zc4h2 유전자를 녹아웃시킨 형질전환 동물모델 및 이의 용도
KR20170034438A (ko) * 2014-08-14 2017-03-28 프라운호퍼-게젤샤프트 츄어 푀르더룽 데어 안게반텐 포르슝에.파우. 통증 치료에서의 cyp2j2 길항제

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KR20130094255A (ko) * 2012-02-15 2013-08-23 고려대학교 산학협력단 신경교세포 생성 조절에 관여하는 fam19a5의 약제학적 용도
KR20170034438A (ko) * 2014-08-14 2017-03-28 프라운호퍼-게젤샤프트 츄어 푀르더룽 데어 안게반텐 포르슝에.파우. 통증 치료에서의 cyp2j2 길항제
KR20160101786A (ko) * 2015-02-17 2016-08-26 울산대학교 산학협력단 Ｆａｍ１９ａ５ 단백질을 포함하는 비만 예방 또는 치료용 조성물 및 이를 이용한 비만 치료제의 스크리닝 방법
KR20160143913A (ko) * 2015-06-04 2016-12-15 충남대학교산학협력단 Zc4h2 유전자를 녹아웃시킨 형질전환 동물모델 및 이의 용도

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