CN117363659A - Method for accurately identifying germ cell xenograft and chimera of Bostrichthys sinensis - Google Patents

Method for accurately identifying germ cell xenograft and chimera of Bostrichthys sinensis Download PDF

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CN117363659A
CN117363659A CN202311653496.8A CN202311653496A CN117363659A CN 117363659 A CN117363659 A CN 117363659A CN 202311653496 A CN202311653496 A CN 202311653496A CN 117363659 A CN117363659 A CN 117363659A
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vasa
bostrichthys sinensis
receptor
germ cells
piwi
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CN117363659B (en
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刘威
董梦丹
张弯弯
易梅生
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Sun Yat Sen University
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Abstract

The invention discloses a method for accurately identifying the germ cell xenograft and chimera of Bostrichthys sinensis, which establishes a technical method for the germ cell transplantation of the Bostrichthys sinensis germ cell living fluorescent marker and receptor Bostrichthys sinensis larva fish, and fills the blank of the prior art; furthermore, the identification method can identify whether the germ cells of the fixed-planted Bostrichthys sinensis exist in the gonad of the recipient fish with high sensitivity. The invention also discloses a primer for identifying the chimera, which has species specificity and can accurately identify the spermary germ cell chimera of the Bostrichthys sinensis.

Description

Method for accurately identifying germ cell xenograft and chimera of Bostrichthys sinensis
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for accurately identifying a germ cell xenograft and chimera of a Chinese bostrichthys sinensis.
Background
Fish farming makes an important contribution to the continuous supply of world quality animal proteins; the method comprehensively utilizes methods of genetics, cell biology, biotechnology and the like to develop biological breeding of aquatic animals such as fishes and the like, creates novel aquatic germplasm with high yield, high quality, disease resistance and stress resistance, and can powerfully promote important ways of transformation, upgrading and green development of modern fishery. The germplasm is the genetic material that the organism's parent transmits to the progeny. In mature individuals of higher animals, germ cells are the only cells that can transfer the carried genetic material to the offspring, and are the cell carriers for alternate germplasm generations. The essence of germplasm creation and improvement is to select and improve germ cells to obtain haploid gamete cells with excellent properties.
Chinese Odontobutis obscura (wall.) kuntzeBostrychus sinensis) Belonging to the family of the snakehead and the genus of the Odontobutis, the snakehead is a rare cultured fish in southeast Asia, has fresh and delicious meat, high nutritive value, large male individuals and high growth speed, and is popular in a large bay area. However, in the artificial culture environment, partial male Odontobutis obscurus can generate the phenomenon of spermary abortion or facultative gonad, resulting in the loss of germplasm resources; even in the case of male fish with normal gonad development, sperm cannot be collected by artificial extrusion in the breeding season. Therefore, the current Bostrichthys sinensis breeding industry generally adopts a natural mating mode to reproduce offspring, and the mode needs to consume a great deal of manpower and material resources and has low offspring yield. Therefore, obtaining a large number of the Chinese bostrichthys sinensis sperms is a key for solving the problem of seed shortage bottleneck of the development of the Chinese bostrichthys sinensis industry.
The fish germ cell transplanting technology, commonly called "abdominal fetuses", refers to transplanting germ cells of a donor fish to an unclosed genital ridge region of a recipient fish and embedding the germ cells into the genital ridge to form a germ line chimeric body and generate gametes or offspring derived from the donor. In 2003 Takeuchi et al first transplanted rainbow trout germ cells into cherry trout, which produced mature gametes and offspring of rainbow trout. Then, the fish germ cell transplantation technology has been successfully applied to other fishes, including carp germ stem cells to goldfish, loach germ stem cells to zebra fish, gobio rarus germ stem cells to zebra fish and gobio scophthalmus maximus germ stem cells to paralichthys olivaceus, so as to obtain carp, loach, gobio rarus and mature gametes of turbot from donor fish sources.
However, there is no report in the prior art on the use of fish germ cell transplantation technology to obtain mature gametes of Bostrichthys sinensis.
The fish germ cell transplanting technology comprises the following two links: firstly, preparing a germ line chimeric body, and secondly, identifying the germ line chimeric body to determine the transplanting condition.
The preparation of the germ line chimera comprises the following 3 steps (1) and (2) are not in sequence): (1) efficiently separating and purifying germ cells of a donor; (2) selecting a suitable receptor; (3) the donor germ cells are transplanted into the recipient's genital ridge and colonize, develop, and mature in the recipient to form a germ line chimera.
In the prior art, researchers transplant donor fish germ cells to the germ ridge of a recipient fish through fluorescent protein dye marks of cell membranes, and the proportion of chimeras carrying fluorescent protein marks is generally 50-90% 1 month after transplantation; however, due to immune rejection, cell cycle and differences in reproduction and differentiation, the germ cells of the donor fish are gradually cleared when transplanted into the xenogeneic recipient fish, and the proportion of germ line chimeras is significantly reduced: after 7 months of transplantation, the chimera proportion was reduced to less than 20%.
Furthermore, as the number of cell divisions, pigmentation and tissue internalization increased, the fluorescent signal in the chimera gradually disappeared, and was hardly observed 2 months after the transplantation, and it was difficult to determine the transplantation condition.
Therefore, based on the above, accurate identification of germ line chimeras is also an important part of the fish germ cell transplantation technology.
Currently, methods for identifying fish germ line chimeras are mainly by using genomic DNA markers (including microsatellite markers and mitochondrial DNA markers) of the donor and recipient to identify the presence or absence of donor fish DNA in the recipient. However, this method has obvious drawbacks: since the genomic DNA sequences of somatic and germ cells are identical, this detection method cannot be used to determine whether the amplified DNA fragment must be derived from a germ cell. In contrast, there is a clear difference in gene transcription levels in different cells, and germ cells have specifically expressed genes. According to the theory, chinese patent CN114592075A discloses a detection method of germ cell xenograft and chimeric gonad after transplantation, wherein the detection method utilizes two PCR amplification to detect the germ cell specific marker gene of the finless eelsdazl3' utr (untranslated region) of Monopteri albi, detecting finless eel germline chimera. However, because the sequences of specific marker genes of fish germ cells have obvious differences, the molecular markers of different germ line chimeras are often not universal, and the molecular markers need to be developed and designed aiming at specific species so as to realize accurate and efficient identification of germ line chimeras. In addition, the detection method needs to utilize two rounds of PCR amplification to detect whether finless eel germ cells exist in gonads of the receptor fish, and is complex in operation and long in time.
Disclosure of Invention
The invention aims to provide a method for accurately identifying the germ cell xenograft and chimera of the Bostrichthys sinensis.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
The invention provides a method for xenograft of germ cells of Bostrichthys sinensis, which comprises the following steps:
(1) Selecting marmoreus fries with the genital ridges not closed as receptors;
(2) Obtaining testis germ cells of the Bostrichthys sinensis serving as a donor; then treating the testis germ cells by using fluorescent dye to obtain the testis germ cells marked by fluorescent protein;
(3) Microinjection of fluorescent protein-labeled testis germ cells into the genital ridges of the receptor, namely transplanting the fluorescent protein-labeled testis germ cells into the small fish of marmoreus to form a chimera of the small fish of marmoreus.
Odontobutis obscura (Ling.) kuntzeOxyeleotris marmorata) Although the snakehead is freshwater fish belonging to the family of the snakehead, the snakehead is better similar to the snakehead in sexual maturity period and physiological characteristics of sperms and ova, and the snakehead is bigger in individual, so that sperms can be discharged through the extrusion abdomen; experiments prove that the chimaera of the spermary germ cells of the Bostrichthys sinensis can be successfully prepared by taking the Bostrichthys marmoratus fries as a receptor.
In the prior art, technicians judge the position of the recipient genital ridge by experience when carrying out donor transplantation, so the problem of inaccurate position judgment often occurs; in addition, the positions of the genital ridges of different fishes are obviously different, so that the position of the genital ridge of the receptor is necessary to be accurately positioned by using a technical means.
Therefore, in the step (1), the invention further utilizes an immunofluorescence method to determine the genital ridge position of the marmoreus larva fish (receptor).
As a specific embodiment of the present invention: in the step (1), fixing the small fish of the marmoreus with 4% paraformaldehyde, and cleaning for 3 times by using a Phosphate Buffer Solution (PBS); then, the solution is permeabilized with Triton X-100 with a volume fraction of 5% for 5min and washed with PBS for 3 times; adding 2% sheep serum, blocking for 1h at room temperature, adding vasa antibody, and incubating overnight; washing 3 times with 0.2% Tween 20, incubating the fluorescent protein-labeled secondary antibody for 1h at room temperature, and washing 3 times with PBS; finally, observing the germ cell condition by using a fluorescence microscope to image, and determining the position of the germ ridge of the fish fry of the marmoreus.
The treatment target of the existing fish immunofluorescence technology is mature ovum or embryo not exceeding 5 days after fertilization, and is generally permeabilized for more than 1 hour by adopting PBS solution of 0.2-0.5% Triton X-100, and then antibody is added for incubation to carry out subsequent experiments (see Spector D L. Immunofluorescence Localization of Nuclear proteins Cold Spring Harbor Protocols, 2011; and HeJ, et al, coded white-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae, nature Protocol, 2020). After embryo development is finished, the individual growth speed of the fish fry is extremely high, the skin and muscle tissues of the fish fry are thickened quickly, young fish with the thickness of more than 7 days after the fish fry are generally subjected to membrane forming, 0.2-0.5% Triton X-100 can hardly penetrate the muscle tissues unless the fish fry are treated for more than 24 hours, but the problems that tissues are damaged after long-time treatment, fluorescent signals cannot be generated and the like can be solved. Therefore, triton X-100 is generally recognized in the art as a permeabilizing agent that is not useful as a sample of larger fish; in other words, no report has been made in the prior art that immunofluorescence techniques were successfully applied to fish bodies.
However, the invention is discovered to be treated by 5% Triton X-100 for 5 minutes after a series of growths and in the occasional case, so that the tissue can be well penetrated and protected, and the signal of the germ cell specific marker gene in the gonad of the fish fry of the Odontobutis obscurus after 10-40 days after fertilization can be detected, so that a good experimental result is obtained. The invention is based on the detection of germ cell specific genes by integral immunofluorescencevasaThe expression of the Odontobutis obscurus genital ridge is defined to be positioned at 1-3 body joints behind the swimming bladder, an important basis is provided for accurate germ cell transplantation, and the proportion of the obtained chimera is improved.
As a further embodiment of the invention, in the step (3), the condition of germ cells in the transplanted marjoram receptor is detected by using an immunofluorescence method, and the method can detect the conditions of the donor germ cells and the receptor germ cells simultaneously; the specific method is the same as the immunofluorescence method in the step (1), but the treated (experimental) object is the transplanted marjoram receptor.
In the step (2), the testis germ cells of the Bostrichthys sinensis are obtained by adopting a conventional method, for example: a method for sorting fluorescent protein by using flow cells to specifically label germ cells, a method for sorting fluorescent protein by using flow cells or magnetic beads to specifically label germ cells, and a method for separating fish germ cells by using density gradient centrifugation. As a specific embodiment of the invention, the density gradient method is adopted in the step (2) to separate and purify the testis germ cells of the Bostrichthys sinensis.
The invention also provides a method for precisely identifying the Chinese bostrichthys sinensis testis germ cell chimera, which comprises the following steps:
extracting total RNA from a marmoreus receptor after transplanting the spermary germ cells of the Chinese marmoreus, and performing reverse transcription to obtain cDNA; detecting whether the Bostrichthys sinensis exists in the receptor by using the cDNA as a template and performing PCR amplificationvasaAnd/orpiwiFragments; if the Bostrichthys sinensis exists in the receptorvasaAnd/orpiwiThe fragment is a schwann-tail testis germ cell chimera;
wherein, the Bostrichthys sinensis is detectedvasaThe primer pair of (a) is as follows:
bsvasaF: CGAAGAGTAACAACCACCAAACTAC（SEQ ID NO:1），
bsvasaR: TGGCTTTTCTCCAGCATCATTCG（SEQ ID NO:2）；
detection of Bostrichthys sinensispiwiThe primer pair of (a) is as follows:
bspiwiF: TGTGTTCTTGAATTTTCCATCAGTATTTAGT（SEQ ID NO:3），
bspiwiR:AGTACACAGCATAATAGCAGACTCATAA（SEQ ID NO:4）。
for the identification of chimeras, the present invention first determined the genes specifically expressed by germ cells in donor Bostrichthys sinensis and acceptor Bostrichthys marmoreus, as there was no germ cell specific DNA but mRNA specifically expressed by germ cellsvasaAndpiwi. In order to distinguish the Bostrichthys sinensis from the Bostrichthys marmoratusvasaAndpiwi mRNA, the invention designs a species specific primer according to the weak sequence difference of homologous genes of two species, and the primer realizes specific amplification of the germ cells of the Bostrichthys sinensisvasaAndpiwimRNA sequence without amplifying the germ cells of marmoreusvasaAndpiwimRNA sequence, accurately identifying whether the fixed-planted spermary germ cells of the Bostrichthys sinensis exist in gonads of the recipient fish.
As a specific embodiment of the invention, the marmoreus receptor 15 days to 9 months after the implantation of the spermary germ cells of the marmoreus is identified.
As a further embodiment of the present invention, the detection of the presence or absence of marmoreus in the recipient is increasedvasaAnd/orpiwiFragments, for comparison, confirm that the amplified sample is or contains gonad tissue of marmoreus, indicating that donor germ cells are indeed chimeric to the gonad of marmoreus; specifically: detecting whether the maculosa marmoreus exists in a receptor by using the cDNA as a template through PCR amplificationvasaAnd/orpiwiFragments;
wherein, the detection of the marmoreusvasaThe primer pair of (a) is as follows:
omvasa F: AAGAGTCCCATCTACCAATAGTACTGT（SEQ ID NO:5），
omvasaR: GGTTTTTCTCCAGCGTCACCCA（SEQ ID NO:6）；
detection of Odontobutis obscurapiwiThe primer pair of (a) is as follows:
ompiwiF: CATCGCGTGTTCTTAATAGCGCCA（SEQ ID NO:7），
ompiwiR: CACAGCATGATAGCGGACTCGTAT（SEQ ID NO:8）。
the invention also provides a primer for identifying the Chinese bostrichthys sinensis testis germ cell chimera, which comprises the steps of detecting the Chinese bostrichthys sinensisvasaAnd/orpiwiPrimer pairs of the fragments; wherein,
detection of Bostrichthys sinensisvasaThe primer pair of the fragment is as follows:
bsvasaF: CGAAGAGTAACAACCACCAAACTAC（SEQ ID NO:1），
bsvasaR: TGGCTTTTCTCCAGCATCATTCG（SEQ ID NO:2）；
detection of Bostrichthys sinensispiwiThe primer pair of the fragment is as follows:
bspiwiF: TGTGTTCTTGAATTTTCCATCAGTATTTAGT（SEQ ID NO:3），
bspiwiR:AGTACACAGCATAATAGCAGACTCATAA（SEQ ID NO:4）。
the invention identifies the germ cell chimera of the eleotris sinensis testisThe primer of (2) also comprises the step of detecting the marmoreusvasaAnd/orpiwiPrimer pairs of the fragments; wherein,
detection of Odontobutis obscuravasaThe primer pair of the fragment is as follows:
omvasa F: AAGAGTCCCATCTACCAATAGTACTGT（SEQ ID NO:5），
omvasaR: GGTTTTTCTCCAGCGTCACCCA（SEQ ID NO:6）；
detection of Odontobutis obscurapiwiThe primer pair of the fragment is as follows:
ompiwiF: CATCGCGTGTTCTTAATAGCGCCA（SEQ ID NO:7），
ompiwiR: CACAGCATGATAGCGGACTCGTAT（SEQ ID NO:8）。
compared with the prior art, the invention has the following advantages:
1. based on the prior art, no report on the abnormal transplanting of the germ cells of the Bostrichthys sinensis exists, the invention establishes a technical method for transplanting the germ cells of the Bostrichthys sinensis by using the vital fluorescent markers of the germ cells of the Bostrichthys sinensis and the receptor Bostrichthys marmoratus, fills the blank of the prior art, and can be applied to the artificial culture industry of the Bostrichthys sinensis;
the method used by the invention is simple, does not need special instruments, and has the advantages of simplicity and high efficiency;
in addition, the invention is beneficial to the development of the 'abdominal reproduction' technology of the Bostrichthys sinensis and is suitable for the detection of the mature gametes of the Bostrichthys sinensis in the heterogeneous production.
2. According to the invention, the position of the genital ridge of the receptor is accurately positioned by utilizing an immunofluorescent protein technology in the process of preparing the chimera for the first time, and then donor germ cell transplantation is carried out;
the invention can reach better experimental results after a series of growths and the treatment of 5% Triton X-100 for 5 minutes under the accidental condition; germ cell specific gene detection based on whole immunofluorescencevasaThe expression of the Odontobutis marmorata genital ridge is accurately positioned, an important theoretical basis is provided for accurate germ cell transplantation, and the proportion of the obtained chimera is also improved.
3. The book is provided withThe invention screens and identifies the specific expression genes of the germ cells of the donor Bostrichthys sinensis and the acceptor Bostrichthys marmoreusvasaAndpiwiand thus based on the acceptor and donorvasaAndpiwithe specific molecular markers of the receptor and the donor fish are respectively designed, the target fragment is amplified in the genital cells of the Bostrichthys sinensis by PCR, and whether the genital cells of the fixed-planted Bostrichthys sinensis exist in the gonad of the receptor fish is identified with high sensitivity. It is particularly notable that even though very few donor germ cells may be present in the chimeric gonads, the present invention allows for sensitive screening of the chimeric by only one round of PCR amplification.
4. The invention relates to a method for preparing a feed for Bostrichthys sinensis and a feed for Bostrichthys marmoratusvasaAndpiwi specific expression of mRNA, designing a primer specific to species, and precisely identifying the spermary germ cell chimera of the Bostrichthys sinensis.
Drawings
FIG. 1 determination of the genital ridge position of Odontobutis marmorata;
FIG. 2 Odontobutis sinensis germ cell transplantation; (a) fluorescent observations of recipient fish 1 day after transplantation; (B) And (C) detecting the expression of a specific marker gene VASA of the germ cells of the Bostrichthys sinensis one month after transplantation for immunofluorescence so as to determine the change of the germ cell number in the Bostrichthys marmoratus before and after transplantation; wherein (B) is statistics of the number of GFP-positive, i.e., VASA-positive cells detected by immunofluorescence in C, (C) is a picture of representative individuals in the transplanted group and the control group, grey arrows indicate recipient-derived germ cells, and white arrows are donor-derived germ cells;
FIG. 3 is a schematic representation of a Bostrichthys sinensis and a Bostrichthys marmoreusvasaAndpiwithe cDNA sequence alignment of (2) and the locus of the designed species-specific primer;
FIG. 4 is a graph of Hua Wu Tangli and Odontobutis obscuravasaAndpiwiwherein the left panel shows their distribution in different tissues of Bostrichthys sinensis and Bostrichthys marmoreus, heart (H), liver (L), spleen (S), brain (B), gill (G), ovary (O) and testis (T); right side view isvasaAndpiwirespectively carrying out electrophoresis patterns on amplification products of 8 tail Chinese bostrichthys sinensis and 8 tail martrichthys marmoreus gonad cDNA;
FIG. 5 is a graph showing the distribution of RFP signals in the receptor of Bostrichthys marmoreus at various times after the germ cell transplantation of Bostrichthys marmoreus and the electrophoresis pattern of PCR products of gonad samples thereof; wherein, (a) is a graph 15 days after the transplantation, (B) is a graph 1 month after the transplantation, (C) is a graph 2 months after the transplantation, (D) is a graph 4 months after the transplantation, and (E) is a graph 8 months after the transplantation.
Detailed Description
The following examples are only for illustration of the invention, and the scope of the invention is not limited to the following examples. The object of the present invention can be achieved by those skilled in the art based on the above disclosure of the present invention and the ranges taken by the parameters.
Example 1
The method for xenograft of the germ cells of the Bostrichthys sinensis comprises the following steps:
1. determining the genital ridge position of the receptor marmoreus fries:
odontobutis marmorata fries (whose genital ridges have not been occluded at this time) 10 days after fertilization were fixed overnight at 4℃with 4% paraformaldehyde, washed 3 times with PBS, permeabilized 5min at room temperature with 5% Triton X-100 in PBS, washed 3 times with PBS, blocked 1h with 2% sheep serum (volume fraction) in PBS at room temperature, and incubated overnight with vasa antibody (abcam, 1:200). The next day was washed 3 times with PBS 0.2% Tween 20, incubated 1h with Green Fluorescent Protein (GFP) labeled secondary antibody (1:400) at room temperature, washed 3 times with PBS, and then imaged using a fluorescence microscope to determine where the genital ridge was located.
As shown in fig. 1 (boxes show the positions of the genital ridges), GFP-positive germ cells are distributed in discrete points in the area of 1-3 individual sections near the spine behind the swimming bladder of marmorus marmoratus, namely the genital ridges of marmorus marmoreus larvae.
2. Obtaining the spermary germ cells of the Bostrichthys sinensis and marking with fluorescent protein:
1) Isolation and purification of testis germ cells (refer to the method of chinese patent CN112608887a for details): the testis was isolated from 5 month old Bostrichthys sinensis, sterilized in 70% ethanol for 30 seconds, washed 3 times with PBS, sheared with medical scissors, added with 1 ml testis digest, and digested at 37deg.C on a 60 rpm shaker for 1 h. The digestive juice comprises the following components: adding 4 mg/mL type IV collagenase, 0.05% DNase I (mass fraction), 0.25% pancreatin and 10% FBS into the L-15 culture medium;
2) Fluorescent protein labeling: after incubating the testis single cells with 200. Mu.l of dye pKH26 (sigma, stock diluted 1:200) for 15 minutes at room temperature, RFP-labeled Bostrichthys sinensis cells were obtained.
3. Transplanting the sperm cell of the Chinese strongylus sinensis:
microinjection of RFP-labeled cells into the genital ridges of the receptor marmoreus larvae treated in the step 1, namely, transplanting the RFP-labeled cells into the marmoreus larvae to form chimeras of the spermary germ cells of the marmoreus;
the distribution of RFP signals in recipient fish was observed 1 day after injection, as shown in fig. 2 a and table 1, significant RFP signals were observed at the genital ridge of more than 95% of recipient fish fries, and more than 85% of recipients were observed after one month of transplantation.
According to the immunofluorescence method in step 1, immunofluorescence experiments were also performed on the marmoreus fries 30 days after transplantation, and the change in germ cell numbers in the transplanted group and the control group (i.e., the recipients not subjected to transplantation) was detected. As shown in B in fig. 2, GFP-markers in the genital ridges of post-transplant recipients compared to control groupvasaThe number of positive cells, namely germ cells, is obviously increased; as shown in C in fig. 2, the increased cells are derived from transplanted donor germ cells, wherein GFP signal (indicated by white arrows) from donor-derived germ cells is slightly weaker than the brightness (indicated by gray arrows) of recipient germ cells.
Comparative example 1
The method for xenograft of the germ cells of the Bostrichthys sinensis comprises the following steps:
1. determining the genital ridge position of the receptor marmoreus fries:
the fish fry of Odontobutis marmorata 10 days after fertilization was fixed overnight with 4% paraformaldehyde at 4 ℃, washed 3 times with PBS, permeabilized 1h at room temperature with 0.5% Triton X-100 in PBS, washed 3 times with PBS, blocked 1h with 2% sheep serum (volume fraction ratio) in PBS at room temperature, and incubated overnight with vasa antibody (abcam, 1:200). The following day was washed 3 times with PBS 0.2% Tween 20, incubated 1h with Green Fluorescent Protein (GFP) labeled secondary antibody (invitrogen, 1:400) at room temperature, washed 3 times with PBS, and no GFP signal was observed under a fluorescence microscope and no fluorescence was observed.
2. Obtaining the spermary germ cells of the Bostrichthys sinensis and marking with fluorescent protein:
the specific procedure was the same as in example 1, and RFP-labeled Bostrichthys sinensis cells were obtained finally.
3. Transplanting the sperm cell of the Chinese strongylus sinensis:
since the genital ridge position of the receptor cannot be accurately located by GFP signal in step 1 of this comparative example, this comparative example microinjects RFP-labeled cells to the vicinity of the genital ridge of the receptor marmoreus larvae, i.e., the vicinity of the swim bladder of the larvae, by virtue of the prior experience; observing the distribution of RFP signals in recipient fish 1 day after injection showed that only 70% of the recipients observed clear RFP signals and that the proportion of individuals containing RFP signals was reduced to 40% 1 month after implantation (see table 1).
Comparative example 2
The method for xenograft of the germ cells of the Bostrichthys sinensis comprises the following steps:
1. determining the genital ridge position of the receptor marmoreus fries:
the fish fry of Odontobutis marmorata 10 days after fertilization was fixed overnight with 4% paraformaldehyde at 4 ℃, washed 3 times with PBS, permeabilized 3 h at room temperature with 0.2% Triton X-100 in PBS, washed 3 times with PBS, blocked 1h with 2% sheep serum (volume fraction ratio) in PBS at room temperature, and incubated overnight with vasa antibody (abcam, 1:200). The following day was washed 3 times with PBS 0.2% Tween 20, incubated 1h with Green Fluorescent Protein (GFP) labeled secondary antibody (invitrogen, 1:400) at room temperature, washed 3 times with PBS, and no GFP signal was observed under a fluorescence microscope and no fluorescence was observed.
2. Obtaining the spermary germ cells of the Bostrichthys sinensis and marking with fluorescent protein:
the specific procedure was the same as in example 1, and RFP-labeled Bostrichthys sinensis cells were obtained finally.
3. Transplanting the sperm cell of the Chinese strongylus sinensis:
also, since the genital ridge position of the receptor could not be accurately located by GFP signal in step 1 of this comparative example, this comparative example uses the prior experience to microinjection of RFP-labeled cells near the genital ridge of the receptor marmoreus larvae, i.e., near the region of the swim bladder of the larvae; observing the distribution of RFP signals in recipient fish 1 day after injection showed that only 50% of the recipients observed clear RFP signals and that the proportion of individuals containing RFP signals was reduced to 20% 1 month after implantation (see table 1).
TABLE 1 proportion of RFP-Positive individuals in the recipient Odontobutis marmorata 1 day and 30 days after the transplantation
The results in Table 1 show that the immunofluorescence method adopted to accurately locate the genital ridge position of the receptor can improve the proportion of the obtained chimera; this is because transplanted donor cells are germ cells that are not viable for long periods of time in non-gonadal regions, and prior art techniques only empirically determine the recipient's genital ridge location, often with errors. Therefore, the invention provides the method for determining the genital ridge position of the receptor by using the immunofluorescence method, provides an important basis for germ cell transplantation, and has remarkable significance for the development of fish germ cell transplantation technology.
Example 2
1. Identifying the specific marker genes of the germ cells of the Bostrichthys sinensis and the Bostrichthys marmoratus:
and respectively extracting total RNA of tissues of the Bostrichthys sinensis and the Bostrichthys marmoreus by using TRIZOL, and performing reverse transcription to obtain cDNA. Adult tissues include heart (H), liver (L), spleen (S), brain (B), gill (G), ovary (O), and testis (T).
By transcriptome sequencing, the obtained Chinese black tail and the obtained marmorusvasaAndpiwithrough sequence alignment, screening and searching for the difference sites (see figure 3) of the cDNA sequences of the two, and designing primers, wherein the specific details are shown below;
odontobutis sinensisvasaThe primer pair is as follows:bsvasaf: CGA AGA GTA ACA ACC ACC AAA CTA C, andbsvasaR： TGG CTT TTC TCC AGC ATC ATT CG；
odontobutis obscura (Willd.) kultzvasaThe primer pair is as follows:omvasaf: AAG AGT CCC ATC TAC CAA TAG TAC TGT, andomvasaR：GGT TTT TCT CCA GCG TCA CCC A；
odontobutis sinensispiwiThe primer pair is as follows:bspiwif: TGT GTT CTT GAA TTT TCC ATC AGT ATT TAG T, andbspiwiR：AGT ACA CAG CAT AAT AGC AGA CTC ATA A；
odontobutis obscura (Willd.) kultzpiwiThe primer pair is as follows:ompiwif: CAT CGC GTG TTC TTA ATA GCG CCA, andompiwiR：CAC AGC ATG ATA GCG GAC TCG TAT。
PCR amplification detection Using the above cDNA as a templatevasaAndpiwiis detected by agarose gel electrophoresisvasaAndpiwiis described herein). As shown in FIG. 4, the Bostrichthys sinensis and the Bostrichthys marmoratusvasaAndpiwiare specifically expressed in the testis and ovary; the invention judges the Chinese bostrichthys sinensis and the marmorus as well known in the art to express in the germ cells onlyvasaAndpiwialso specifically expressed in germ cells.
Further, in this example, odontobutis sinensis and Odontobutis martensi were performedvasaAndpiwithe species-specific amplification of (2) was performed by using 4 pairs of primers from cDNA of the testis of 8-tail Bostrichthys sinensis and 8-tail Bostrichthys marmoratus, respectively, and the results showed thatbsvasaThe primer pair can only amplify a single fragment from the cDNA of 8 tail Bostrichthys sinensis, and no fragment product is detected in the cDNA of the testis of 8 tail Bostrichthys marmoratus; converselyomvasaThe primer pair can amplify a single fragment only from the cDNA of 8 tail marmoreus, but no cDNA of the testis of 8 tail marmoreus is detectedAny fragment product. Also, the process of the present invention is,bspiwiandompiwithe primer pair of (2) also has good species specificity.
2. Identifying the spermary germ cell chimera of the Bostrichthys sinensis by using the primers:
randomly selecting the marchanna maculosa receptor 15 days to 9 months after transplantation in the example 1, observing the receptor by a fluorescence microscope (see an example of fig. 5), extracting total RNA of the receptor, performing reverse transcription to obtain cDNA, and detecting whether the marchanna maculosa and the marchanna maculosa are in the receptor by PCR by using the primers respectivelyvasaAndpiwi。
as shown in FIG. 5, in the Odontobutis martensii receptor 15 days after transplantation, a significant RFP signal was observed in the abdomen of most individuals, and the PCR results showed that 9/10 of the samples were detectablebsvasaAndbspiwifragments, all samples being detectableomvasaAndompiwithe fragment, therefore, had a 90% chimeric rate at 15 days post-implantation. The fluorescence signal of the recipients at 1, 2, 4 and 9 months after the transplantation gradually weakens, and the RFP signal is basically difficult to see at 2 months after the transplantation, namely, the chimera is difficult to judge at the moment; however, the ratio of the chimeras was clearly judged by PCR to be about 90%, 70% and 16.7%, respectively (chimeric gonad ratio = calculated as the percentage of positive gonad number of detected chimeric bostrichthys sinensis germ cells to the number of samples detected).
The results of this example demonstrate that the identification method of the present invention allows accurate screening of chimeras, and that chimeras can still be sensitively screened by only one round of PCR amplification, with very few donor germ cells likely to be present in the chimeric gonads.
Furthermore, as is clear from the results of the present example,bsvasa、bspiwi、omvasaandompiwithe primer pairs of the present invention have good species specificity, so the present inventionbsvasaPrimer pairbspiwiOne or more primer pairs are selected, or simultaneously selected fromomvasaPrimer pairompiwiOne or more primer pairs are selected, so that accurate identification of the spermary germ cell chimera of the Bostrichthys sinensis can be realized.
The present invention may be summarized in other specific forms without departing from the spirit or essential characteristics thereof. The above-described embodiments of the present invention are to be considered in all respects only as illustrative and not restrictive. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments according to the essential technology of the present invention fall within the scope of the present invention.

Claims (6)

1. A method for xenograft of germ cells of Bostrichthys sinensis is characterized by comprising the following steps:
(1) Selecting a small fish of marbled fish with a not-closed genital ridge as a receptor, and determining the genital ridge position of the receptor by using an immunofluorescence method;
the immunofluorescence method comprises the following steps: fixing the marbled fish receptor with 4% paraformaldehyde, and cleaning with phosphate buffer solution for 3 times; then, performing permeabilization treatment for 5min by using Triton X-100 with the volume fraction of 5%, and washing for 3 times by using phosphate buffer solution; adding 2% sheep serum, sealing at room temperature for 1 hr, addingvasaIncubating the antibody overnight; washing 3 times with 0.2% Tween 20, incubating the fluorescent protein-labeled secondary antibody for 1h at room temperature, and washing 3 times with PBS; finally, observing germ cells by utilizing a fluorescence microscope to image, thereby determining the position of a germ ridge of a receptor of the marmorus marmoratus;
(2) Obtaining testis germ cells of the Bostrichthys sinensis serving as a donor; then treating the testis germ cells by using fluorescent dye to obtain the testis germ cells marked by fluorescent protein;
(3) Microinjection of fluorescent protein-labeled testis germ cells into the genital ridges of the receptor, namely transplanting the fluorescent protein-labeled testis germ cells into the small fish of marmoreus to form a chimera of the small fish of marmoreus.
2. The method according to claim 1, wherein in the step (3), germ cells in the transplanted recipient are detected by immunofluorescence.
3. The method for precisely identifying the Chinese bostrichthys sinensis testis germ cell chimera prepared by the method of claim 1, which is characterized by comprising the following steps:
extracting total RNA from a marmoreus receptor after transplanting the spermary germ cells of the Chinese marmoreus, and performing reverse transcription to obtain cDNA; detecting whether the Bostrichthys sinensis exists in the receptor by using the cDNA as a template and performing PCR amplificationvasaAnd/orpiwiFragments; if the Bostrichthys sinensis exists in the receptorvasaAnd/orpiwiThe fragment is a schwann-tail testis germ cell chimera;
wherein, the Bostrichthys sinensis is detectedvasaThe primer pair of (a) is as follows:
bsvasa F: CGAAGAGTAACAACCACCAAACTAC，
bsvasa R: TGGCTTTTCTCCAGCATCATTCG；
detection of Bostrichthys sinensispiwiThe primer pair of (a) is as follows:
bspiwi F: TGTGTTCTTGAATTTTCCATCAGTATTTAGT，
bspiwi R:AGTACACAGCATAATAGCAGACTCATAA。
4. the method according to claim 3, wherein the presence or absence of Odontobutis marmorata in the subject is detectedvasaAnd/orpiwiFragments, for comparison; specifically: detecting whether the maculosa marmoreus exists in a receptor by using the cDNA as a template through PCR amplificationvasaAnd/orpiwiFragments;
wherein, the detection of the marmoreusvasaThe primer pair of (a) is as follows:
omvasa F: AAGAGTCCCATCTACCAATAGTACTGT，
omvasa R: GGTTTTTCTCCAGCGTCACCCA；
detection of Odontobutis obscurapiwiThe primer pair of (a) is as follows:
ompiwi F: CATCGCGTGTTCTTAATAGCGCCA，
ompiwi R: CACAGCATGATAGCGGACTCGTAT。
5. identification of the chimeric germ cells of the testis of Bostrichthys sinensis obtained by the method of claim 1The primer of the body is characterized by comprising the following steps of detecting the Bostrichthys sinensisvasaAnd/orpiwiPrimer pairs of the fragments; wherein,
detection of Bostrichthys sinensisvasaThe primer pair of the fragment is as follows:
bsvasa F: CGAAGAGTAACAACCACCAAACTAC，
bsvasa R: TGGCTTTTCTCCAGCATCATTCG；
detection of Bostrichthys sinensispiwiThe primer pair of the fragment is as follows:
bspiwi F: TGTGTTCTTGAATTTTCCATCAGTATTTAGT，
bspiwi R:AGTACACAGCATAATAGCAGACTCATAA。
6. the primer set forth in claim 5, further comprising detecting marmoreusvasaAnd/orpiwiPrimer pairs of the fragments; wherein,
detection of Odontobutis obscuravasaThe primer pair of the fragment is as follows:
omvasa F: AAGAGTCCCATCTACCAATAGTACTGT，
omvasa R: GGTTTTTCTCCAGCGTCACCCA；
detection of Odontobutis obscurapiwiThe primer pair of the fragment is as follows:
ompiwi F: CATCGCGTGTTCTTAATAGCGCCA，
ompiwi R: CACAGCATGATAGCGGACTCGTAT。
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