CN114934075A - Screening method and application of major gene in zebra fish heart development related gene cluster deletion mutant - Google Patents
Screening method and application of major gene in zebra fish heart development related gene cluster deletion mutant Download PDFInfo
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Abstract
The invention discloses a screening method and application of major genes in zebra fish heart development related gene cluster deletion mutants, which comprises the steps of determining target positions of hoxb1b, hoxb5b, hoxb6b and hoxb8b genes in a zebra fish hoxbb gene cluster, respectively designing target gRNA sequences and primers for PCR amplification, carrying out in vitro transcription to obtain gRNA and Cas9mRNA, carrying out microinjection on the gRNA and the Cas9mRNA into zebra fish, and hybridizing the zebra fish with mutation with a wild type to obtain a heterozygote F 1 Sexual maturity of F 1 The homozygous mutant is obtained by mating, the gene hoxb1b with obvious phenotype is preliminarily screened by carrying out microimaging and single gene identification on the offspring subjected to gene knockout, the single cell sequencing data is used for analyzing the early expression condition of four single genes in the hoxb and carrying out in-situ hybridization, gene knockdown and overexpression to screen out the main effect gene hoxb1b related to the early development of the heart, and the method is favorable for searching pathogenic targets and screening drugs.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a screening method and application of major genes in zebra fish heart development related gene cluster deletion mutants.
Background
The CRISPR/Cas technology is a gene editing system for comparing fire and heat in recent years after ZFIN and TELEN, and has been widely applied to various modes of organisms in the world based on the advantages of simplicity, high efficiency, easiness in operation and the like. Two CRISPR/Cas9 action systems were mainly found in the early stages of the study: form I and form III. With the progress and development of scientific technology, two doctors from the university of california at berkeley and university of omson collaborated with 2012 to discover a type ii CRISPR/Cas9 system. This system is simple in construction, and only requires RNA at both ends and an endonuclease of Cas9 to perform a cleavage function to a target site. Later, two laboratories, Zhang Feng from Mazhou Jumbo and George Church from Harvard medical college, acted the Cas9 system simultaneously on human cells and generated efficient cleavage. CRISPR/Cas9 was then successively deployed in laboratories around the world and successfully deployed in individual model animals. Work on gene knockout mediated by CRISPR/Cas9 has been implicated from bacteria to fish to primates. The process carries out site-directed knockout on species at each layer of biological evolution stage, provides an effective technical means for researching organism gene function and biological evolution, and the CRISPR/Cas becomes an effective tool for developing gene editing and constructing a disease model.
In the process of genome evolution and environment adaptation of organisms, some genes can form a series of repeated genes to form a gene cluster. In animals and plants and in microorganisms, the occurrence of tandem repeat events brings about an increase in the copy number of genes and allelic variation in the genome of the organism. For the research of complex functional tandem repeat sequence-tandem repeat gene, the formation mechanism or evolution rule of important character and phenotype related gene/gene family of organism can be revealed. The commonly used research method in recent years is to determine the key components and genetic rules generated by specific traits through methods such as phenotypic observation, genetics, metabolome, transcriptome and resequencing, and then to finally determine the key genes through positioning and cloning key candidate genes. The above method takes a long time and is expensive. The method for constructing the mutant by the CRISPR technology and screening the major gene by observing the phenotype and verifying the molecular biology is efficient, quick and strong in operability.
The Hox gene is a transcription factor family which regulates the body morphology construction during early embryo development. When the Hox gene is mutated, it often results in malformation of early embryonic development. Hox gene has been doubled in the whole genome in the evolution process, the drosophila which is found at first only contains one Hox gene cluster, and the quadruped animals such as mice have been doubled in the whole genome twice to form 4 Hox gene clusters, and most teleost fishes such as zebra fish have been doubled in the genome once to form 7 Hox gene clusters, and 48 gene members in total. Currently, the research on the Hox gene function is mainly the role in the construction of animal body morphology, but part of the Hox gene plays an important role in organ development, for example, Hox10 plays a certain role in the development process of intestines and kidneys, Hoxb1 shows the phenomenon of abnormal development of the heart in mammals, and the Hox gene is reported to be expressed in second heart domain (SHF) precursor cells or Neural Crest Cells (NCC). In 2014, Steven E Weicksel et al studied hoxb1b and hoxb1a and found that they together regulate the development of hindbrain rhombohedral nodes. In mice, Hoxb1 was studied mainly for its regulatory role in the pharyngeal arch and for its effects on cardiac SHF and OFT development. Joshua s.waxman et al studied the function of zebrafish by knocking down hoxb5b in 2008. However, the mechanism by which Hox genes regulate cardiac precursor cell development is not known.
In patients with heterozygote loss of the HOXB cluster, various organ abnormalities including abnormalities of the heart, brain and face, among which significant heart disease such as ventricular septal defect, have occurred, studies have shown that the HOXB cluster plays a role in normal cardiac development, but no specific genes critical to this cluster have been identified. Furthermore, these studies have not advanced the potential pathways downstream of HoxB, nor provided a mechanistic link to explain abnormal morphology, and the downstream pathways of the hox gene during cardiac development and maturation are still unclear. In the early stage, a mutant with a large fragment of deleted hoxbb cluster is established in zebra fish, abnormal heart forms such as heart failure, heart circulation defect and the like are observed after 5 days of embryo fertilization, but few studies on the hoxbb gene cluster at home and abroad at the early stage can not explain a genetic regulation mechanism of abnormal heart development caused by the deletion of the hoxbb gene cluster, and the major effective site is not identified all the time. The method for screening the major genes has the advantages of high efficiency, rapidness, safety and economy, and also provides an application prospect for accurate administration of target genes of future congenital heart diseases.
Disclosure of Invention
The invention mainly aims to provide a screening method of major genes in zebra fish heart development related gene cluster deletion mutants, which utilizes a model organism of zebra fish to perform gene knockout on four genes of hoxb1b, hoxb5b, hoxb6b and hoxb8b in a hoxb gene cluster through a CRISPR technology, and utilizes molecular biology and biological information technology to screen out major genes which play a role in heart development in the hoxb cluster.
The invention also aims to provide a zebra fish hoxb1b gene knockout mutant, which designs a specific targeting site by using CRISPR/Cas9 technology for the first time to realize the specific knockout of the hoxb1b gene in zebra fish.
The invention further aims to provide application of the zebrafish hoxb1b gene knockout mutant as an animal model in hoxb1b gene biological function analysis, disease modeling or target drug screening.
The purpose of the invention is realized by the following technical scheme:
the invention provides a screening method of major genes in zebra fish heart development related gene cluster deletion mutants, which comprises the following steps:
s1, determining target positions of four genes including hoxb1b, hoxb5b, hoxb6b and hoxb8b in a zebra fish hoxbb gene cluster, and respectively designing target gRNA sequences;
s2, designing and synthesizing gRNA upstream primers and gRNA downstream primers of the four target spots respectively;
s3, performing PCR amplification by respectively using the gRNA upstream primer and the gRNA downstream primer by using the gRNA framework plasmid as a template;
s4, in-vitro transcription is carried out on the PCR product of the step S3, and the gRNA of hoxb1b, hoxb5b, hoxb6b and hoxb8b is obtained through purification;
s5, injecting the purified gRNA and Cas9mRNA into a unicellular zebra fish embryo in a micro-injection mode respectively, and extracting DNA for T7E1 enzyme detection after 24 hours;
s6, after the zebra fish is sexually mature, hybridizing the zebra fish with mutation with wild zebra fish to obtain heterozygote F of the gene types of hoxb1b, hoxb5b, hoxb6b and hoxb8b 1 Heterozygote F 1 Identification of genotype after adult fish ligation transformation, F after sexual maturation 1 Mating to obtain homozygous mutant;
s7, carrying out microimaging on offspring subjected to successful knockout of hoxb1b, hoxb5b, hoxb6b and hoxb8b genes, carrying out single gene identification, and screening genes with obvious phenotypes according to images corresponding to homozygous mutants with different single-site deletions;
s8, analyzing the sequencing results of the zebra fish in 10hpf, 14hpf and 18hpf single cells respectively, screening genes which are obviously expressed in early heart development, simultaneously screening the in situ hybridization expression conditions of probes (hoxb1b, hoxb5b, hoxb6b and hoxb8b) which are combined with different genes, and screening the main effective gene in the hoxb cluster which is hoxb1b by contrast with the result of the step S7.
Preferably, in step S1, the sequences of the target gRNAs of the hoxb1b, hoxb5b, hoxb6b and hoxb8b genes are shown in SEQ ID NO: 1-4, respectively.
Preferably, in step S2, the gRNA upstream primer is T7+ Target site + gRNA forward primer, and is the primers T7-hoxb1b-sfd, T7-hoxb5b-sfd, T7-hoxb6b-sfd and T7-hoxb8b-sfd with the sequences shown in SEQ ID NO. 5-8, respectively, and the gRNA downstream primer is shown in SEQ ID NO. 9.
Preferably, in step S4, the sequences of the gRNAs of hoxb1b, hoxb5b, hoxb6b and hoxb8b are shown in SEQ ID NOS: 11-14, respectively.
Preferably, in step S5, the detection primers used for the enzyme detection of T7E1 include upstream primer hoxb1b-F and downstream primer hoxb1b-R with sequences shown in SEQ ID NO. 15 and SEQ ID NO. 16, upstream primer hoxb5b-F and downstream primer hoxb5b-R with sequences shown in SEQ ID NO. 17 and SEQ ID NO. 18, upstream primer hoxb6b-F and downstream primer hoxb6b-R with sequences shown in SEQ ID NO. 19 and SEQ ID NO. 20, and upstream primer hoxb8b-F and downstream primer hoxb8b-R with sequences shown in SEQ ID NO. 21 and SEQ ID NO. 22.
The invention also provides a zebra fish hoxb1b gene knockout mutant which is obtained by a screening method of the major gene hoxb1b related to early development of the heart of zebra fish, wherein the zebra fish hoxb1b gene knockout mutant generates a pericardial cavity swelling and heart cyclization abnormality phenotype.
The invention also provides application of the zebra fish hoxb1b gene knockout mutant as an animal model in hoxb1b gene biological function analysis, disease modeling or target drug screening.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention constructs a mode for discovering major genes in a gene cluster to search key genes in a plurality of genes in the gene cluster, the hoxb1b gene knockout generates mutation phenotypes such as abnormal cardiac cyclization, no obvious cardiac deformity is observed in other hox gene cluster gene knockout, and the screened hoxb1b is the major gene in the hoxb cluster related to early cardiac development, thereby having important significance for the pathogenic mechanism research and the drug screening of subsequent targets.
(2) Compared with the traditional gene knockout technology, the CRISPR/Cas9 technology has the characteristics of low toxicity, high accuracy, high efficiency, short success period and the like, the invention designs a section of specific targeting site by using the CRISPR/Cas9 technology for the first time to realize the specific knockout of the hoxb1b gene in the zebra fish, the hoxb1b gene encodes 229 amino acids in total, compared with a mutant fish line with large deletion of hoxbb fragments, the zebra fish hoxb1b knockout mutant which is obtained by deleting 52 bp of the mutant codes for 52 amino acids, deleting 4bp of the mutant codes for 107 amino acids, deleting 5bp of the mutant codes for 51 amino acids and deleting 8bp of the mutant codes for 50 amino acids (figure 2), has more direct biological significance of a single gene and can provide a specific target spot for later-stage drug screening, the gene knockout model animal model can provide theoretical basis for the research of congenital heart related diseases.
(3) Research results show that Hoxb1b mainly plays a role in regulating cardiac development in cardiac progenitor cells and differentiation processes, different subdomains expressed by Hoxb1, Hoxa1 and Hoxa3 in SHF contribute to development of atrial and cardiac outflow tract (OFT) of mice, and the expression of transcription factors such as gata5 and hand2 in mutants and a control group is obviously different through research on expression of the transcription factors important in the cardiac development process in Hoxb1b, and the phenomenon is further confirmed through analyzing the change of the expression level of the genes by in situ hybridization. In addition, the dual-luciferase reporter gene shows that hoxb1b has no significant effect on hand2 promoter, but gata5 inhibits the hand2 promoter, so that hoxb1b can indirectly inhibit the expression of hand2 in zebra fish through gata 5. The zebra fish HOXB1b gene knockout mutant constructed by the invention can be used for researching the influence of hox gene on early development of heart and research mechanism thereof, is beneficial to researching downstream gene regulation network thereof, has good reference significance for HOXB-deleted diseases, and provides scientific basis and thought for analyzing, treating and preventing related diseases.
(4) The hoxb1b gene mutation can be stably inherited, and the hoxb1b gene mutation can be stably inherited -/- Mutant zebrafish of different mutation types all have homozygous lethality, about 11dpf (day post fertilization) all die, the phenotype is serious, and the abnormalities of obvious morphological and physiological indexes such as pericardial cavity swelling, abnormal cardiac cyclization and the like are observed at 5dpf (figure 5).
Drawings
FIG. 1 shows the target positions of zebrafish hoxb1b, hoxb5b, hoxb6b and hoxb8b genes and the T7E1 enzyme digestion detection gel map.
FIG. 2 shows truncated proteins mutated by the mutations of zebrafish hoxb1b and hoxb5 b.
FIG. 3 is a graph showing the results of screening key genes in the hoxbb cluster; a: the section with hoxb1b and hoxb5b are highlighted by a schematic drawing of single cell sequencing data. b: stage 14hpf, single cell transcriptional profiling and in situ hybridization of hoxb1b, hoxb5b, hoxb6b and hoxb8 b. The results showed that only hoxb1b and hoxb5b were expressed in cardiac mesoderm. c hoxb5b compared to control -/- Neither the mutant nor the hoxb1b MO injected wild type embryos showed cardiac defects at 5dpf, scale bar 0.2 mm; thus eliminatinghoxb5b, results showed that only hoxb1b was the key candidate for the hoxb cluster.
FIG. 4 is the determination of key genes in the hoxbb cluster; a-d: pictures of in situ hybridization of hoxb1b, hoxb5b, hoxb6b and hoxb8b at 4.5 dpf; e-f': hoxb1b -/- Mutants showed pericardial enlargement and failure of cardiac cyclization at 5 dpf; g-g': wild type embryos injected with hoxb1b MO showed cardiac defects at 5 dpf; h-h': injection of hoxb1b mRNA (50pg) was overexpressed in wild type embryos, resulting in cardiac abnormalities at 5 dpf.
FIG. 5 is hoxb1b -/- Compared with the control group, the mutant has an abnormal phenotype of pericardial cavity enlargement at 5dpf, marks the position of the abnormal phenotype by "+" and scales to 1000 um.
FIG. 6 shows the phenotype of the hoxb1b mutant with morphological and physiological abnormalities of cardiac development; a-c: hoxbb at 5dpf compared to control -/- And hoxb1b -/- (ii) a phenotypic abnormality of the heart; d-e: the relative blunt cardiac cyclization angle of the hoxb and hoxb1b mutants compared to control; f-g: ventricular systolic and diastolic hoxb1b -/- Ventricular volume increased at 6dpf compared to control; *: p<0.05，**：P<0.01，***：P<0.001。
FIG. 7 shows that hoxb1b indirectly inhibits hand2 expression in zebrafish through gata 5; a-b: qPCR detection and in situ hybridization showed a down-regulation of gata5 expression in the hoxbb and hoxb1b mutants compared to controls; c: the dual-luciferase reporter gene analysis shows that hoxb1b activates the expression of a gata5 promoter and has a remarkable effect, n is greater than 3; d-e: qPCR detection and in situ hybridization showed that hand2 expression was up-regulated in the hoxb1b mutant compared to the control; f: dual luciferase reporter assay showed that gata5 inhibited hand2 promoter, n >3, asterisks in the graph indicate statistical significance, assessed by one-way anova: p <0.01,.: p < 0.001; AC: digestive tract, BA: gill bow, OFT: outflow tract, AVC: the atrioventricular compartment.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
Examples
1. Material and apparatus
1.1 Fish for experiments
The zebra fish used in the experiment is derived from a zebra fish platform of the aquatic product and life college of Shanghai ocean university.
1.2 plasmids
In vitro synthetic Cas9mRNA plasmids used for the experiments were from the literature: liu D, Wang Z, Xiao A, Zhang Y, Li W, Zu Y, Yao S, Lin S, Zhang B. effective gene targeting in zebrafish mediated by a zebrafish-code-optimized case 9 and evaluation of off-targeting effect.J Gene genomics.2014 Jan 20; 41(1) 43-6.gRNA backbone plasmids were derived from the literature: chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ. genome editing with RNA-guided Cas9 nucleic in zebrafish embroys, Cell Res,2013,23 (4): 465-472.
1.3 Primary reagents
DNA Clean&Contentitor-5 (ZYMO RESEARCH, D4004), general DNA purification kit (TIANGEN, DP204-03),
t7 in vitro Transcription Kit (Ambion, AM1314), ethanol (absolute ethanol) (Chemicals group, Inc., 10009218), Premix Taq TM (Ex Taq TM Version 2.0plus dye)(TAKARA，RR902)，DNA Marker I(TIANGEN，MD101-02)，T7endonuclease 1(NEW ENGLAND
M0302L), Rapid plasmid Mini kit (TIANGEN, DP105), DH5a competent cells (Tiangen Biochemical technology Co., Ltd., CB101-03), 2BeasyTaq PCR Supermix (+ dye) (TAKARA, AS111-12), LB Broth (Shanghai Biotech., D915KA6602), LB BrothBroth agar (Shanghai Producer, D911KA6566), pMD TM 19-T Vector Cloning Kit(TAKARA，6013)。
1.4 Main Instrument
PCR apparatus (brand: BIO-RAD, model: c1000 Touch) TM Thermal Cycler), small centrifuge (brand: eppendorf, model: centrifuge 5424), shaker mixer (brand: VORTEX-GENIE, model: G560E), ultraviolet spectrophotometer (brand: thermo Scientific, model: nanodrop 2000C), electrophoresis apparatus (brand: BIO-RAD, type: PowerPac Basic), photographic glue apparatus (brand: Bio-Rad, model: gel Doc EZ Imager), electronic balance (brand: METTLER TOLEDO, type: AL104), glass capillary (brand: WPI, model: TW100F-4), Milli-Q Direct 8 ultra pure water system (brand: millipore, type: Milli-Q Direct 8), vertical pin puller (brand: NARISHIGE, type: PC-10), constant temperature shaker (brand: innova, type: 40R), needle grinder (brand: NARISHIGE, model number: EG-400), a micro syringe pump (brand: WARNER, model: PLI-100A), thermostat water bath (brand: fine macro, type: h1401438, DK-8D), 4 ℃ refrigerator (brand: haier, type: HYC-610), -a low temperature refrigerator at 40 ℃ (brand: haier, type: DW-40L508), -80 ℃ ultra low temperature refrigerator (brand: pana-sonic, model: MDF-U53V), autoclave (brand: SANYO, type: MLS-3780).
2. Designing gRNA knockout target point and two-side primers of each gene in gene cluster
1) Principle of target site design
The first principle is that the gRNA knockout site can destroy important gene sites to disable a target gene, and meanwhile, the gRNA knockout site is designed strictly according to the design principle of gRNA, and detection primers are simple when the knockout result is detected.
2) Target site design
Http through the website: // www.genome-engineering. org/criprpr/, hoxb1b, hoxb5b, hoxb6b and hoxb8b Cas9 target prediction is carried out on the target gene, and some targets are selected for later use.
3) Target site specific detection
Comparing the found target with the zebra fish genome through a Blast page of an NCBI (national center for Biotechnology information) website, further detecting the specificity of the standby target, redesigning a new target site if a sequence which is the same as the target site appears in other positions of the genome, and obtaining four target gRNA sequences as target sites
hoxb1b：GGCTTTTCCGTCGTGAATAT(SEQ ID NO:1)
hoxb5b：AAATCCGAGTCAGGAGACCC(SEQ ID NO:2)
hoxb6b：GGAAGTCTGGAAGACTCGC(SEQ ID NO:3)
hoxb8b：GGCAGACCATCTGTGCTGTA(SEQ ID NO:4)。
4) Primer design for four target spots
The knock-out efficiency was observed using the T7E1 enzyme to detect mutations, and the following points were noted in designing primers:
a. the difference in distance between the upstream and downstream primers and the target is preferably greater than 100bp,
b. the distance between the two primers and the target point is preferably more than 100bp,
c. the length of the PCR product is preferably 350-500 bp.
Since the transcription kit is special for the T7 promoter, the gRNA upstream primer F1
Namely TAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC；
The sequence of primer T7-hoxb1b-sfd is
The sequence of primer T7-hoxb5b-sfd is
The sequence of primer T7-hoxb6b-sfd is
The sequence of primer T7-hoxb8b-sfd is
The sequence of a downstream primer R1(trans reverse), i.e., a gRNA reverse primer, is
5’-AAAAAAAGCACCGACTCGGTGCCAC-3’(SEQ ID NO:9)。
3. Synthetic gRNA
3.1 Synthesis of gRNA sequences and detection primers
The hoxb1b gRNA has the sequence
TAATACGACTCACTATAGGCTTTTCCGTCGTGAATATGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:11)。
The hoxb5b gRNA sequence was
TAATACGACTCACTATAAAATCCGAGTCAGGAGACCCGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:12)。
The hoxb6b gRNA has the sequence
TAATACGACTCACTATAGGAAGTCTGGAAGACTCGCGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:13)。
The hoxb8b gRNA has the sequence
TAATACGACTCACTATAGGCAGACCATCTGTGCTGTAGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:14)。
The primer sequences for detecting mutation by the T7E1 enzyme in step S5 are respectively
The upstream primer hoxb1 b-F: TAGTTACATTCCCGAGGGACG, respectively; (SEQ ID NO:15)
The downstream primer hoxb1 b-R: TTCGTCTTGGAGTCGCTGTC, respectively; (SEQ ID NO:16)
The upstream primer hoxb5 b-F: TAACGGCGCTATGAACGCTT, respectively; (SEQ ID NO:17)
The downstream primer hoxb5 b-R: TGTGGAGTTTGACCGTCGTT; (SEQ ID NO:18)
The upstream primer hoxb6 b-F: AGGGGTCGTCAAACCTACAC, respectively; (SEQ ID NO:19)
The downstream primer hoxb6 b-R: CAGAGGTGAGTTCGATGCCT, respectively; (SEQ ID NO:20)
The upstream primer hoxb8 b-F: CCGGATTTCTACCACCACGG; (SEQ ID NO:21)
The downstream primer hoxb8 b-R: CTGCTTTGCTGCTGGGAAAC are provided. (SEQ ID NO:22)
3.2 specific synthetic procedure for gRNA
A. Shake the fungus
B. Extraction of plasmid 14h
C. High fidelity enzymatic PCR
D. Reaction system:
2％Agarose 120V，30min
PCR product purification (DNA Clean & Concentrator-5) method 3-4 tubes mixing one-tube purification
In vitro transcription of MAXiScriptT7 (see in particular MAXiScript) TM Kit Instruction Manual，Ambion)
Reaction system:
heating in 37 deg.C water bath for 80min, adding 1uL TURBO DNase, and heating in 37 deg.C water bath for 15 min.
LiCl/ethanol precipitation
1) Add 2.5. mu.L of 4M LiCl (5M plus 2. mu.L, 7.5M plus 1.5. mu.L), 100. mu.L of 100% ethanol to the above system;
2) incubation at-80 ℃ for >1 h;
3) centrifuging at 12000rpm at 4 deg.C for 15 min;
4) discarding the supernatant, rinsing twice with 70% ethanol pre-cooled on ice;
5) standing at room temperature for about 5min, volatilizing residual ethanol, and dissolving the precipitated RNA with RNase-free water;
6) finally, the concentration is measured by using the Nanodrop and the RNA quality is detected by electrophoresis.
4. Synthesis of Cas9mRNA
For details, see Protocol of
T7Ultra Kit, briefly as follows:
A. carrying out amplification culture on the stored Cas9 plasmid bacterial liquid, and shaking the bacteria for about 16 h;
B. extracting plasmid, measuring concentration, and electrophoretically detecting plasmid quality
C.XbaI digestion of linearized plasmid
Enzyme digestion system:
the reaction was carried out at 37 ℃ for 30 min.
D.1% Agarose gel electrophoresis 110v, 30min to determine whether the enzyme digestion is complete
E. Purification of
Purification procedures refer to DNAClean & Concentrator-5, ZYMO RESEARCH kit.
MmessagemMACHINE T7Ultra kit in vitro transcription
The reaction conditions refer to the specification, and the whole experimental process is carried out in an Rnase-free environment.
Reaction system:
oscillating, mixing uniformly, centrifuging, carrying out water bath at 37 ℃ for 2-3 h, then adding 1uL DNase, mixing uniformly, carrying out water bath at 37 ℃ for 15min, and removing the DNA template.
F. + PolyA tail
1) Reaction system:
2) 2.5uL of the mixture was reserved as a control
3) Mixing the above mixture with +4uL E-PAP, and gently mixing
4) Heating in water bath at 37 deg.C for 45min
5) 1% gel electrophoresis 150v, 20min for observing tailing (1uL gel loading Buffer +1uL RNA)
Cas9mRNA purification by aqueous LiCl precipitation
The experimental steps are as follows:
Precipitation of RNA from Solution at a Concentration≥400ng/μl
1) adding 50uL LiCl into the Cas9mRNA added with the A tail, and uniformly mixing;
2) precipitation at-80 ℃ for >1 h;
3) centrifuging at 14000rpm for 15min at 4 ℃;
4) discarding the supernatant, washing the precipitate with 70% ethanol pre-cooled on ice for 2 times;
5) with an appropriate amount of RNase-free H 2 And dissolving the O.
H. Quality detection
And (4) measuring the product concentration by using the Nanodrop, and detecting whether the RNA band size is correct and the RNA quality is qualified or not by electrophoresis.
Cas9mRNA and gRNA microinjection and knockout efficiency detection
Cas9mRNA and gRNA are prepared into mixed liquid according to a certain proportion, under the general condition, the Cas9mRNA is 300-600 pg, the gRNA is 80-100 pg, and the mixed liquid is injected into a zebra fish one-cell stage animal pole. The same pair of fish spawning injections required some uninjected eggs to be left for control in the knockout assay.
T7E1 enzyme digestion detection
The specific experimental procedures refer to the instructions of the T7E1 endonuclease kit.
A. Extracting genome by an alkaline lysis method. Taking 3 groups of embryos with normal phenotype after 48hpf injection, adding 5 eggs in each group, 50uL of 50mM NaOH, and oscillating at 95 ℃ for 10 min; again 95 ℃ for 10min, then add 5uL of 1M Tris.HCl (pH 8.0) to neutralize the NaOH, 12000rpm/min, centrifuge for 6 min.
PCR system:
after 34cycles, 2% Agarose 120V was run for 30 min.
And C, detecting the T7E1 endonuclease, and if the PCR product has a single band, performing enzyme digestion detection.
Enzyme digestion system:
the reaction conditions are as follows:
95℃30s
85℃5min
4℃∞
after the reaction, 0.25uL of T7E1 enzyme was added in a water bath at 37 ℃ for 45 min.
D. Electrophoresis of enzyme-digested products
2% agarose electrophoresis 120V, 30 min. Generally, low voltage electrophoresis is used, so that the size of the restriction enzyme digestion band can be better distinguished, and whether the restriction enzyme digestion band is the target band or not can be confirmed, so as to judge whether the knockout is successful or not, as shown in fig. 1.
6. Screening of major genes
6.1 in situ hybridization and photography of 4 genes in Gene Cluster, evaluation of major genes affecting cardiac development in Gene Cluster by Gene expression location
1) And (3) probe synthesis: designing probes for synthesizing hoxb1b, hoxb5b, hoxb6b and hoxb8b, inserting the amplified probe sequences into an expression vector PXT7 plasmid for sequencing, after determining the insertion direction, linearizing the plasmid or amplifying a probe sequence carrying a T7 promoter by PCR, and after homozygously obtaining a target fragment, transcribing in vitro.
2) Fixing the embryo: wild-type zebrafish embryos of 14hpf were selected and fixed with 4% paraformaldehyde overnight at 4 ℃ and subsequently subjected to in situ hybridization experiments.
3) The whole course of the in situ hybridization experiment requires 3 days
Day 1:
A. rehydrating, taking out fixed embryo from refrigerator at-20 deg.C,
b.4% paraformaldehyde for 20 min.
C. Hybridization of
Firstly, pre-hybridization is carried out, embryos are placed in hybridization solution without probes, the hybridization solution is placed in a65 ℃ hybridization furnace for 2-3 h, then the probes are added, and hybridization is carried out in the 65 ℃ hybridization furnace for about 14 h.
the samples were washed, eluted into PBST, and blocked.
And (3) blocking the sample by using blocking solution containing 1% of lamb serum and 2% of BSA (bovine serum albumin), wherein the blocking time is generally 2-3 h.
After blocking, the anti-digoxin antibody was added at a ratio of 1:3000 and shaken overnight at 4 ℃.
Day 3:
the sample was taken out from the shaker at 4 ℃ and washed with 1 XPBST for 6X 15min, and then developed in the dark with a developing solution prepared from NBT-BCIP.
After the color development is finished, the reaction is stopped by 1 XPBST, 5 XPBST 1min, 1 XPBST, finally put into 1 XPBST, the embryo is placed in 3% methyl cellulose for photographing.
4) Photographing and storing, as shown in fig. 4, a-d: pictures of in situ hybridization of hoxb1b, hoxb5b, hoxb6b, and hoxb8b at 4.5 dpf; e-f': hoxb1b -/- Mutants showed pericardial enlargement and hearts at 5dpfFailure of dirty cyclization; g-g': wild type embryos injected with hoxb1b MO showed cardiac defects at 5 dpf; h-h': injection of hoxb1b mRNA (50pg) was overexpressed in wild type embryos, resulting in cardiac abnormalities at 5 dpf.
6.2. Using data and codes reported in the literature, highly enriched genes were compared to known cell type markers and single cell sequencing results pictures were generated, to see if the genes of interest were expressed at this stage, and key genes hoxb1b were screened by analyzing the results in combination with in situ hybridization results in the previous step.
qPCR experiment
qPCR reaction system (20 μ L system):
placing the sample in
Experiments are carried out in the instrument, and after data are subsequently analyzed, graph plotting and significance analysis are carried out by utilizing GraphPad software.
8. Confocal microscopy imaging and analysis
Laser scanning confocal microscope for WT and hoxbb at 5dpf -/- And hoxb1b -/- The zebra fish embryos are photographed and stored; the angle of cardiac cyclization was measured using ImageJ (NIH, USA).
Individual 5dpf embryos were placed laterally on glass pressure drop slides. Embryonic heart physiological data were recorded using a zeiss Axioplan microscope and a high speed digital camera in germany. The ventricular diastolic area and systolic area were measured using ImageJ (NIH, USA).
The data were plotted and analyzed for significant differences using GraphPad software (fig. 6).
9. Dual luciferase reporter gene assay
HEK293T cells were cultured in 24-well cell culture plates and used
HD transfection reagents were used for transfection. Each well was transfected with 50ng or 100ng of reporter gene vector and different gradients of gene expression vector. Detection was performed 36 hours after transfection using a Dual-Luciferase Reporter System (Dual-Luciferase Reporter Assay System). Each set of experimental biology was repeated three more times and each experiment was performed in parallel with three wells. The results of the experiment were examined using t-test and plotted using GraphPad 6.0 and analyzed for significant differences (fig. 7).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Shanghai ocean university
Screening method and application of major gene in <120> zebra fish heart development related gene cluster deletion mutant
<141> 2022-06-30
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggcttttccg tcgtgaatat 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaatccgagt caggagaccc 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggaagtctgg aagactcgc 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggcagaccat ctgtgctgta 20
<210> 5
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
taatacgact cactataggc ttttccgtcg tgaatatgtt ttagagctag aaatagc 57
<210> 6
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
taatacgact cactataaaa tccgagtcag gagacccgtt ttagagctag aaatagc 57
<210> 7
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
taatacgact cactatagga agtctggaag actcgcgttt tagagctaga aatagc 56
<210> 8
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
taatacgact cactataggc agaccatctg tgctgtagtt ttagagctag aaatagc 57
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aaaaaaagca ccgactcggt gccac 25
<210> 11
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
taatacgact cactataggc ttttccgtcg tgaatatgtt ttagagctag aaatagcgga 60
cagattcatg tcctggacgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120
<210> 12
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
taatacgact cactataaaa tccgagtcag gagacccgtt ttagagctag aaatagcgga 60
cagattcatg tcctggacgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120
<210> 13
<211> 119
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
taatacgact cactatagga agtctggaag actcgcgttt tagagctaga aatagcggac 60
agattcatgt cctggacgtt atcaacttga aaaagtggca ccgagtcggt gcttttttt 119
<210> 14
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
taatacgact cactataggc agaccatctg tgctgtagtt ttagagctag aaatagcgga 60
cagattcatg tcctggacgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tagttacatt cccgagggac g 21
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ttcgtcttgg agtcgctgtc 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
taacggcgct atgaacgctt 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tgtggagttt gaccgtcgtt 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aggggtcgtc aaacctacac 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cagaggtgag ttcgatgcct 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ccggatttct accaccacgg 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ctgctttgct gctgggaaac 20
Claims (8)
1. The screening method of the major gene in the zebra fish heart development related gene cluster deletion mutant is characterized by comprising the following steps:
s1, determining target positions of four genes including hoxb1b, hoxb5b, hoxb6b and hoxb8b in a zebra fish hoxbb gene cluster, and respectively designing target gRNA sequences;
s2, designing and synthesizing gRNA upstream primers and gRNA downstream primers of the four target spots respectively;
s3, performing PCR amplification by respectively using the gRNA upstream primer and the gRNA downstream primer by using the gRNA framework plasmid as a template;
s4, in-vitro transcription is carried out on the PCR product of the step S3, and the gRNA of hoxb1b, hoxb5b, hoxb6b and hoxb8b is obtained through purification;
s5, injecting the purified gRNA and Cas9mRNA into a unicellular zebra fish embryo in a micro-injection mode respectively, and extracting DNA for T7E1 enzyme detection after 24 hours;
s6, hybridizing the zebra fish with mutation with wild zebra fish after the zebra fish becomes sexually mature to obtain heterozygote F of the gene types of hoxb1b, hoxb5b, hoxb6b and hoxb8b 1 Heterozygote F 1 Identification of genotype after adult fish ligation and transformation, F after sexual maturation 1 Mating to obtain homozygous mutant;
s7, carrying out microscopic imaging on offspring successfully knocked out by the hoxb1b, the hoxb5b, the hoxb6b and the hoxb8b genes, carrying out single gene identification, and screening genes with obvious phenotypes according to images corresponding to homozygous mutants with different single site deletions;
s8, analyzing the sequencing results of the zebra fish single cells at 10hpf, 14hpf and 18hpf respectively, screening genes which are obviously expressed in early heart development, combining the in situ hybridization expression conditions of single genes of hoxb1b, hoxb5b, hoxb6b and hoxb8b in the hoxb cluster, and screening a main effective gene in the hoxb cluster, which is hoxb1b, by contrasting with the results of the step S7.
2. The screening method of the major genes in the zebra fish heart development related gene cluster deletion mutant as claimed in claim 1, wherein in step S1, the sequences of the target gRNAs of the hoxb1b, hoxb5b, hoxb6b and hoxb8b genes are respectively shown in SEQ ID NO 1-4.
3. The screening method of the major gene in the zebra fish heart development related gene cluster deletion mutant according to claim 1, wherein in step S2, the gRNA upstream primer is a T7+ Target site + gRNA forward primer, which is a primer T7-hoxb1b-sfd, a primer T7-hoxb5b-sfd, a primer T7-hoxb6b-sfd and a primer T7-hoxb8b-sfd, which are shown in SEQ ID NO. 5-8, respectively, and the sequence of the gRNA downstream primer is shown in SEQ ID NO. 9.
4. The method for screening a major gene in a zebrafish heart development related gene cluster deletion mutant according to claim 1, wherein sequences of gRNAs of hoxb1b, hoxb5b, hoxb6b and hoxb8b in step S4 are shown in SEQ ID NO. 11-14, respectively.
5. The screening method of the major genes in the zebra fish heart development related gene cluster deletion mutant as claimed in claim 1, wherein in step S5, the detection primers used in the T7E1 enzyme detection comprise an upstream primer hoxb1b-F and a downstream primer hoxb1b-R with sequences as shown in SEQ ID NO. 15 and SEQ ID NO. 16, an upstream primer hoxb5b-F and a downstream primer hoxb5b-R with sequences as shown in SEQ ID NO. 17 and SEQ ID NO. 18, an upstream primer hoxb6b-F and a downstream primer hoxb6b-R with sequences as shown in SEQ ID NO. 19 and SEQ ID NO. 20, and an upstream primer hoxb8b-F and a downstream primer hoxb8b-R with sequences as shown in SEQ ID NO. 21 and SEQ ID NO. 22.
6. The method for screening major genes in the zebra fish heart development related gene cluster deletion mutant according to claim 1, wherein in step S8, the zebra fish whole embryo in situ hybridization of target genes combined with probes hoxb1b, hoxb5b, hoxb6b and hoxb8b is analyzed by using single cell sequencing results, the expression conditions of the four genes hoxb1b, hoxb5b, hoxb6b and hoxb8b in the hoxb gene cluster in the heart progenitor cell region are compared and verified mutually, and the heart development abnormal phenotype gene which is expressed in the heart progenitor cell region and is similar to the heart development abnormal phenotype gene after the deletion of the hoxb gene cluster is screened and determined as the key gene hoxb1 b.
7. A zebrafish hoxb1b gene knockout mutant which is obtained by the screening method of a major gene hoxb1b related to the early development of the heart of the zebrafish as claimed in any one of claims 1 to 6 and has a phenotype of generating pericardial cavity enlargement and abnormal cyclization of the heart.
8. The use of the zebrafish hoxb1b knockout mutant as claimed in claim 7 as an animal model for the analysis of biological functions of hoxb1b gene, disease modeling or target drug screening.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105647969A (en) * | 2016-02-16 | 2016-06-08 | 湖南师范大学 | Method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647969A (en) * | 2016-02-16 | 2016-06-08 | 湖南师范大学 | Method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout |
Non-Patent Citations (3)
| Title |
|---|
| NICOLAS BERTRAND等: "Hox genes define distinct progenitor sub-domains within the second heart field", DEV BIOL., vol. 353, no. 2, pages 8 * |
| 张立凤;钟涛;桂永浩;: "外源性视黄酸对斑马鱼心血管系统发育的影响", 中国实验动物学报, no. 02 * |
| 张绪帅: ""CRISPR/Cas9 系统介导的七鳃鳗和斑马鱼基因组编辑方法的建立与优化", 中国优秀硕士学位论文全文数据库 基础科学辑, pages 13 - 19 * |
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