CN114592075A - Method for heterogeneously transplanting ricefield eel germ cells and detecting chimeric gonads after transplantation - Google Patents
Method for heterogeneously transplanting ricefield eel germ cells and detecting chimeric gonads after transplantation Download PDFInfo
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Abstract
The invention belongs to the technical field of aquatic animal genetic breeding, and discloses a method for detecting a rice field eel germ cell xenograft and a chimeric gonad after the transplantation. The invention establishes the fluorescence labeling and flow-type fineness of the original germ cell living body of the finless eelTechnical method for sorting fluorescent cells, preparing receptor fish with germ cell deletion and transplanting germ cells of finless eels in juvenile stage by using cytometer and specifically expressing the germ cells of finless eelsdazlA PCR primer is designed in the 3' -UTR region of the gene, a target segment can be amplified under the condition of few ricefield eel germ cells through two rounds of PCR, and whether the colonized ricefield eel germ cells exist in the gonad of a receptor fish or not is identified with high sensitivity. The method provided by the invention is simple and efficient, has high sensitivity, can be effectively applied to the early detection of producing the ricefield eel gamete by utilizing the 'abdominal reproduction' technology, and has important application prospects in the aspects of the development of the 'abdominal reproduction' technology of the ricefield eels and the solution of the industrial problem of shortage of ricefield eel fries.
Description
Technical Field
The invention belongs to the technical field of aquatic animal genetic breeding, and particularly relates to a method for detecting a chimeric gonad of a rice field eel germ cell xenograft and after the transplantation. The method can be used for detecting the planting success rate of the ricefield eel after the germ cells are transplanted in a heterogeneous mode. Is suitable for the development of the 'reproduction by abdomen' technology of the finless eel and the early detection of the mature gamete of the finless eel produced by different species.
Background
The fish germ cell transplantation technique refers to a technique of implanting germ cells obtained from a donor fish into a recipient fish to obtain a germ line chimeric fish and producing gametes or offspring derived from the donor, and may also be referred to as a fish "reproduction by abdomen" technique. In 2003, japanese researchers successfully transplanted Primordial Germ Cells (PGCs) of rainbow trout (Oncorhynchus mykiss) into the bodies of rainbow trout (Oncorhynchus masou) (Takeuchi et al, 2003), and first achieved "reproduction by abdomen" of fish. In 2006, the separated and purified rainbow trout A-type spermatogonial stem cells (A-SG) are transplanted into allogeneic recipient fish to obtain functional male and female gametes (Okutsu et al, 2006) derived from donor fish, which marks that germ cell transplantation technology obtains important breakthrough. At present, the technology is successful in various freshwater fishes, and carp, loach, gobiocypris rarus and turbot mature gametes from donor fish sources are obtained by transplanting carp reproductive stem cells to goldfish (Franek et al, 2021), loach reproductive stem cells to zebrafish (Saito et al, 2008), gobiocypris rarus reproductive stem cells to zebrafish (Zhang et al, 2021) and turbot reproductive stem cells to flounder (Zhou et al, 2021).
Monopterus albus (Monopterus albus) is an important famous and special-purpose object for aquaculture in China. The sex reversal characteristics of the female and male ricefield eel consubstantiality and the first female and the last male cause that female ricefield eels are generally small in fecundity due to small individuals, the mature male ricefield eels are few, and gametes can be obtained only after the male ricefield eels are dissected to obtain gonads during artificial propagation, so that the artificial propagation of the ricefield eels is difficult and high in cost, the breeding of the ricefield eels mainly depends on wild fishing, but the requirement of the ricefield eel industry on the fries is difficult to meet only depending on the wild fishing, and therefore, the shortage of the fries is a bottleneck which restricts the development of the breeding industry. Transplanting the ricefield eel germ cells into receptor fishes with large fecundity and short sexual maturation period, establishing mature ricefield eel germ cell transplanting technical process, realizing 'reproduction by abdomen' of the ricefield eels, being expected to produce a large amount of ricefield eel eggs or sperms, and further solving the seedling shortage bottleneck of the ricefield eel industrial development.
In the case of the technology of fish germ cell transplantation, firstly, it is necessary to establish an effective isolation technique of germ stem cells of donor fish, and the isolation operation is required to maintain the activity of the germ stem cells in order to obtain donor cells that can be used for transplantation. Secondly, preparing sterile fish with germ cells deleted or triploid as a transplant receptor; finally, there is a need to establish an efficient method for detecting chimeric gonads after transplantation.
The distance between the relative relationship between the donor and the recipient fish determines the success of germ cell transplantation. At present, the fish germ cell transplantation technology is successful among different families, genera and species, but the successful case of fish germ cell transplantation among different orders is not seen yet. In classification, finless eels belong to the order gilling, the family gilling, the genus finless eels, the order gilling are of a small variety of fish, and these fish do not meet the characteristics of large fecundity or short sexual maturation cycle that fish should have as transplant recipients.
Therefore, the problem of transplanting different interocular reproductive cells needs to be solved for developing research on transplanting ricefield eel reproductive cells. In addition, the research on transplanting the reproductive cells of the finless eels needs to separate and obtain the reproductive stem cells of the finless eels with activity, and how to carry out living body marking on the primordial reproductive cells of the finless eels and separate and purify the primordial reproductive cells of the finless eels to obtain the primordial reproductive cells of the finless eels with activity is another problem to be solved. The enzyme digestion damages cells, the enzyme and enzyme concentration used for different species are different, different research papers of the same species have different reports (Goto et al, 2010; Zhang et al, 2020), the rice field eel digestive juice is not reported, and the high-purity primordial germ cells are obtained by separating and purifying the rice field eel primordial germ cells which are fluorescently marked in the embryonic stage on the premise of ensuring the cell activity, which is the difficulty of the invention.
The detection of chimeric gonads after transplantation is also an urgent problem to be solved, and the method for detecting genomic DNA is effective in detecting gametes (generally sperms) of donor fish generated by receptor fish after transplantation, because it can be determined that the donor fish germ cells are really in the receptor fish, otherwise mature gametes cannot be generated, the method is to design primers for detection according to sequence differences of genomic DNA, but the method is not suitable for the detection of chimeric gonads which do not generate mature gametes after transplantation. Because even if genomic DNA fragments of donor fish are detected in chimeric gonads of recipient fish, it cannot be determined that the amplified DNA fragments must be derived from germ cells, and the genomic DNA of other types of cells is the same as that of the germ cells. Germ cells do not have specific genes, but only specifically expressed genes.
The main reasons for detecting genomic DNA in the existing reports may be as follows:
1. for detecting whether gametes generated by the transplanted recipient fish are derived from the donor fish, the genomic DNA can be directly detected, but the method is not suitable for detecting the chimeric gonads which do not generate mature gametes.
2. The detection of genomic DNA from a donor fish in a chimeric gonad allows the determination of cells from the donor fish in the recipient fish, but not the germ cells. Studies using this method for detection do not distinguish germ cells from other cells. Additional experimental manipulations are necessary to distinguish germ cells.
3. There are reports in which the DNA sequence of a gene specifically expressed by a germ cell (e.g., vasa) is detected, and from this, the germ cell of a donor fish is determined in the gonad of a recipient fish, which is not strict (or wrong principle) because the DNA sequences of the gene specifically expressed by a germ cell are the same in different types of cells, and only the mRNA of the gene specifically expressed by a germ cell is detected, and the germ cell can be determined.
At present, no research report or technical data related to the abdominal reproduction of the ricefield eels exist, and the establishment of the method for detecting the chimeric gonads of the ricefield eels after the xenotransplantation of the ricefield eels germ cells has important application prospect.
Disclosure of Invention
The invention aims to provide a method for detecting the xenotransplantation of ricefield eel germ cells and the chimeric gonads after the transplantation, and the invention reports the method for the first time, firstly, a primer is designed according to a 3' -UTR sequence of a dazl gene transcript specifically expressed by the ricefield eel germ cells, and whether the chimeric gonads after the germ cell transplantation contain the ricefield eel germ cells or not is quickly detected through PCR; has the advantages of simplicity, high efficiency and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical measures:
a method for detecting chimeric gonads of ricefield eel germ cells after xenotransplantation and transplantation comprises the following steps:
1) injecting morpholino capable of blocking translation of carp or zebra fish dndmRNA into carp or zebra fish fertilized egg, and taking carp or zebra fish juvenile fish with rejected germ cells as recipient fish of ricefield eel reproductive transplantation;
2) injecting living fluorescence markers to the eel embryos at the 1 cell stage, and selecting the eel embryos with the fluorescence markers; selecting embryo tissues behind yolk, digesting the cut eel embryo tissues by using Ringer balanced salt solution containing 0.05-0.3% of collagenase V and 0.5-2% of sodium citrate, lightly blowing the cut eel embryo tissues by using a 5ml, 10ml or 20ml sterile syringe for 2-10 times at intervals of 10-30 minutes, wherein the total time of enzyme digestion is not more than 2 hours, filtering out undissociated tissue blocks by using a cell sieve with the aperture of 70 mu m or 100 mu m, centrifuging the cell blocks at 2000rpm for 3-8 minutes, re-filtering the cell blocks by using a 40 mu m or 70 mu m cell sieve after re-suspending the cells by using PBS solution containing fetal bovine serum, reserving the filtrate, and observing and confirming whether the filtrate is a single cell suspension with complete appearance under a microscope. Performing fluorescence sorting on the single cell suspension meeting the requirements by using a flow cytometer to obtain GFP positive cells, namely ricefield eel primordial germ cells, injecting the ricefield eel primordial germ cells to gonad primordium positions of carps or zebra fish juvenile fish with endogenous germ cells removed through a glass needle to obtain receptor fish embedded with the ricefield eel germ cells;
3) extracting total RNA of gonads of 1-12 months old recipient carps or zebra fishes after the transplantation of the ricefield eel germ cells, and performing reverse transcription to obtain c DNA;
4) taking the cDNA obtained in the step 3) as a template, and amplifying a dazl gene sequence specifically expressed by the ricefield eel germ cells by nested PCR; primers for the first round of PCR nested PCR were eel-dazl-F1: 5'-agtatgccctcctttctcct-3', eel-dazl-R1: 5'-aattgatgccttgattgtcc-3', respectively; the second round of PCR takes the product of the first round of PCR reaction as a template, and the primer sequences are as follows: eel-dazl-F2: 5'-cagagcaccctcaccatacc-3', eel-dazl-R2: 5'-gaatagccgaaagcccttat-3' are provided.
The finless eel dazl fragment obtained by amplification after the first round of PCR is 744bp, and the finless eel dazl fragment obtained by amplification after the second round of PCR is 355 bp; indicating that the integrated ricefield eel germ cells exist in the gonad of the receptor fish.
In the above method, preferably, the carp morpholino is: 5'-cctgctgtagctgctgtccctccat-3', respectively; the morpholino of zebrafish is: 5'-gctgggcatccatgtctccgaccat-3' is added.
In the above method, preferably, in the PBS containing fetal bovine serum in step 2), the volume ratio of fetal bovine serum to PBS is 2%;
the protection scope of the invention also includes: the method is applied to commercial culture of the finless eels.
Compared with the prior art, the invention has the following advantages and effects:
at present, no method for detecting the xenotransplantation of ricefield eel germ cells and the chimeric gonads after transplantation exists, the invention establishes the technical methods of the living fluorescence labeling of the original ricefield eel germ cells, the sorting of fluorescent cells by a flow cytometer, the preparation of germ cell deletion receptors and the transplantation of the ricefield eel germ cells in the juvenile stage, designs a PCR primer according to a 3' -UTR region of a dazl gene specifically expressed by the ricefield eel germ cells, can amplify a target segment under the condition of few ricefield eel germ cells by two-round PCR, and identifies whether the transplanted ricefield eel germ cells exist in the gonads of the receptors with high sensitivity. The method used by the invention is simple, does not need special instruments, and has the advantages of simplicity and high efficiency. The invention is suitable for the development of the ricefield eel 'reproduction by abdomen' technology and the detection of the heterogeneously produced ricefield eel mature gamete. Aiming at the reported content, the invention solves the following technical difficulties:
1. aiming at the separation of primordial germ cells, on one hand, the applicant selects collagenase V with small damage to cells to treat tissues and gropes the concentration and concentration time of digestive juice enzyme by finless eels; the second aspect is to reduce the time for enzyme treatment by the operation of manual blowing. The blowing step, the applicant tried to blow with a conventional pipette, which takes a long time to digest the finless eel embryonic tissue (in case of blowing 5 times at 20 minutes intervals, it is probably 4-6 hours). When the pipette was replaced with a 10ML syringe blow, the enzyme treatment time could be significantly shortened (within 2 hours of digestion with 5 blows per 20 minutes interval).
2. For the detection after transplantation, because no germ cell specific gene exists and only the germ cell specific expression gene exists, the gene dazl specifically expressed by the germ cell in the finless eel is firstly determined, although the gene dazl is also specifically expressed in the germ cell of the carp and the zebra fish, in order to distinguish the dazl mRNA of the finless eel from the dazl mRNA of the carp and the zebra fish, the sequence difference between the dazl mRNA of the finless eel and the dazl mRNA of the carp and the zebra fish needs to be found, generally, the mRNA sequence similarity of the same gene of different species is higher, and the primer needs to be tried to specifically amplify the dazl mRNA sequence of the finless eel and not to amplify the dazl mRNA sequence of the finless eel or the zebra fish;
since the number of the integrated donor germ cells may be small in the grafted chimeric gonads, in this case, the amount of the extracted donor cell-derived RNA is trace, and the trace amount of mRNA is difficult to detect in the conventional one-round RT-PCR (reverse transcription-PCR), we have designed a two-round RT-PCR method for detection, so that the target fragment is detected in the case of a small number of donor germ cells. The two-round PCR method can detect the target sequence under the condition of small number of the integrated donor germ cells, has high sensitivity, and is suitable for detecting the germ cells integrated in the gonads at various stages after transplantation.
Drawings
FIG. 1 fluorescence labeling and sorting of original germ cell living bodies of finless eel.
FIG. 2 is an electrophoresis diagram of two rounds of PCR products of carp gonad samples after the transplantation of finless eel germ cells.
FIG. 3 is an electrophoresis diagram of two rounds of PCR products of zebra fish gonad samples after the transplantation of ricefield eel germ cells.
Wherein M is DNA Marker, C is finless eel gonad sample as positive control.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
a method for detecting chimeric gonads of ricefield eel germ cells after xenotransplantation and transplantation comprises the following steps:
1) injecting morpholino (5'-CCTGCTGTAGCTGCTGTCCCTCCAT-3', 6ng) capable of blocking translation of carp dndhmin mRNA into carp fertilized eggs, completely eliminating germ cells of carp juvenile fish after the dndhmin is successfully injected, and taking the juvenile fish with the eliminated germ cells as a recipient fish for reproductive transplantation of finless eels.
2) Injecting GFP-nos 3' UTR mRNA 450pg capable of specifically marking original germ cells of the finless eel in living body fluorescence to finless eel embryos in a 1-cell stage, selecting finless eel embryos with fluorescence marks, shearing embryo tissues behind yolk by using dissecting scissors under a stereoscopic microscope, digesting the sheared finless eel embryo tissues by using Ringer balanced salt solution containing 0.1 percent of collagenase V and 1 percent of sodium citrate, lightly blowing the tissue by using a 10ml sterile syringe for 5 times every 20 minutes, ensuring that the total time of enzymatic digestion is not more than 2 hours, filtering out undissociated tissue blocks by using a cell sieve with the aperture of 100 mu m, centrifuging the tissue blocks at 2000rpm for 5 minutes, re-filtering the tissue blocks by using a cell sieve with the aperture of 40 mu m after re-suspending the cells by using 2 percent of FBS/PBS solution, retaining filtrate, and observing and confirming whether the filtrate is single-cell suspension with complete appearance under the microscope. And (2) carrying out fluorescence sorting on the single cell suspension meeting the requirements by using a flow cytometer to obtain GFP positive cells, namely, the cells are ricefield eel primordial germ cells (figure 1), and injecting the ricefield eel primordial germ cells to the gonad primordium of the carp juvenile fish with the endogenous germ cells removed through a glass needle to obtain the receptor fish of the chimeric ricefield eel germ cells.
3) Extracting total RNA of gonads of recipient carps of 12 months after the transplantation of the ricefield eel germ cells, and performing reverse transcription to obtain cDNA.
4) Taking the cDNA obtained in the step 3) as a template, and amplifying a dazl gene sequence specifically expressed by the ricefield eel germ cells through nested PCR; primers for nested PCR were eel-dazl-F1: 5'-AGTATGCCCTCCTTTCTCCT-3', eel-dazl-R1: 5'-AATTGATGCCTTGATTGTCC-3', respectively; eel-dazl-F2: 5'-CAGAGCACCCTCACCATACC-3', eel-dazl-R2: 5'-GAATAGCCGAAAGCCCTTAT-3' are provided.
The nested PCR comprises two rounds of PCR reactions, the first round of PCR takes the total gonadal cDNA in the step 4) as a template, and the primer sequences are as follows: eel-dazl-F15 '-AGTATGCCCTCCTTTCTCCT-3', eel-dazl-R15 '-AATTGATGCCTTGATTGTCC-3', PCR amplification program: 95 ℃ for 3 min; 32cycles (95 ℃, 15 s; 65 ℃, 15 s; 72 ℃, 30 s); 72 ℃ for 5 min. The second round of PCR takes the product of the first round of PCR reaction as a template, and the primer sequences are as follows: eel-dazl-F25 '-CAGAGCACCCTCACCATACC-3', eel-dazl-R25 '-GAATAGCCGAAAGCCCTTAT-3', PCR amplification program: at 95 ℃ for 3 min; 35cycles (95 ℃, 15 s; 65 ℃, 15 s; 72 ℃,20 s); 72 ℃ for 5 min.
5) Agarose gel electrophoresis was performed for the analysis of the results of the PCR reaction products in step 5). : and (3) respectively carrying out electrophoresis on the first round PCR product and the second round PCR product in the step (3) by using 1% agarose gel at the voltage of 200V for 15 minutes, analyzing the electrophoresis result through PCR product bands, wherein the finless eel dazl fragment obtained by amplification after the first round PCR is 744bp, and the finless eel dazl fragment obtained by amplification after the second round PCR is 355 bp. Electrophoresis results show that finless eel specific dazl transcript fragments can be amplified in carp gonads transplanted with finless eel germ cells (the chimeric gonad proportion is 2/23 multiplied by 100 percent and 8.69 percent) (figure 2), and the situation that the finless eel germ cells are integrated in the gonads of recipient carps is shown.
The proportion of the chimeric gonads is calculated according to the percentage of the number of the positive gonads of the detected reproductive cells of the chimeric finless eels in the number of the detected samples.
Example 2:
a method for detecting chimeric gonads of ricefield eel germ cells after xenotransplantation and transplantation comprises the following steps:
1) injecting morpholino (5'-GCTGGGCATCCATGTCTCCGACCAT-3', 6ng) capable of blocking the translation of dandmRNA of the zebra fish into fertilized eggs of the zebra fish, completely eliminating germ cells of the zebra fish larvae after the dnd morpholino is successfully injected, and taking the zebra fish larvae with the germ cells eliminated as recipient fish of the reproductive transplantation of the finless eels.
2) Injecting GFP-nos 3' UTR mRNA 450pg capable of specifically marking original germ cells of the finless eel in living body fluorescence to finless eel embryos in a 1-cell stage, selecting finless eel embryos with fluorescence marks, shearing embryo tissues behind yolk by using dissecting scissors under a stereoscopic microscope, digesting the sheared finless eel embryo tissues by using Ringer balanced salt solution containing 0.1 percent of collagenase V and 1 percent of sodium citrate, lightly blowing the tissue by using a 10ml sterile syringe for 5 times every 20 minutes, ensuring that the total time of enzymatic digestion is not more than 2 hours, filtering out undissociated tissue blocks by using a cell sieve with the aperture of 100 mu m, centrifuging the tissue blocks at 2000rpm for 5 minutes, re-filtering the tissue blocks by using a cell sieve with the aperture of 40 mu m after re-suspending the cells by using 2 percent of FBS/PBS solution, retaining filtrate, and observing and confirming whether the filtrate is single-cell suspension with complete appearance under the microscope. And (3) performing fluorescence sorting on the single-cell suspension meeting the requirements by using a flow cytometer to obtain GFP positive cells, namely the eel primordial germ cells, and injecting the eel primordial germ cells to the gonad primordium of the zebra fish juvenile fish with the endogenous germ cells removed through a glass needle to obtain the receptor fish of the embedded eel germ cells.
3) Extracting total RNA of the sexual gland of the receptor zebra fish of 3 months after the transplantation of the ricefield eel germ cells, and performing reverse transcription to obtain cDNA.
4) Taking the cDNA obtained in the step 3) as a template, and amplifying a dazl gene sequence specifically expressed by the ricefield eel germ cells by nested PCR; primers for nested PCR were eel-dazl-F1: 5'-AGTATGCCCTCCTTTCTCCT-3', eel-dazl-R1: 5'-AATTGATGCCTTGATTGTCC-3', respectively; eel-dazl-F2: 5'-CAGAGCACCCTCACCATACC-3', eel-dazl-R2: 5'-GAATAGCCGAAAGCCCTTAT-3' are provided.
The nested PCR comprises two rounds of PCR reactions, the first round of PCR takes the total gonadal cDNA in the step 4) as a template, and the primer sequences are as follows: eel-dazl-F15 '-AGTATGCCCTCCTTTCTCCT-3', eel-dazl-R15 '-AATTGATGCCTTGATTGTCC-3', PCR amplification program: 95 ℃ for 3 min; 32cycles (95 ℃, 15 s; 65 ℃, 15 s; 72 ℃, 30 s); 72 ℃ for 5 min. The second round of PCR takes the product of the first round of PCR reaction as a template, and the primer sequences are as follows: eel-dazl-F25 '-CAGAGCACCCTCACCATACC-3', eel-dazl-R25 '-GAATAGCCGAAAGCCCTTAT-3', PCR amplification program: at 95 ℃ for 3 min; 35cycles (95 ℃, 15 s; 65 ℃, 15 s; 72 ℃,20 s); 72 ℃ for 5 min.
5) Agarose gel electrophoresis was performed for the analysis of the results of the PCR reaction products in step 5). : and (3) respectively carrying out electrophoresis on the first round PCR product and the second round PCR product in the step (3) by using 1% agarose gel at the voltage of 200V for 15 minutes, analyzing the electrophoresis result through PCR product bands, wherein the finless eel dazl fragment obtained by amplification after the first round PCR is 744bp, and the finless eel dazl fragment obtained by amplification after the second round PCR is 355 bp. Electrophoresis results show that a finless eel specific dazl transcript fragment can be amplified in the gonad of the transplanted finless eel germ cells (the chimeric gonad proportion is 3/13 multiplied by 100 percent and 23.08 percent) (figure 3), and the situation that the finless eel germ cells are integrated in the gonad of the receptor zebra eel is shown.
Sequence listing
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Claims (4)
1. A method for detecting chimeric gonads of ricefield eel germ cells after xenotransplantation and transplantation comprises the following steps:
1) the injection can block carp or zebra fishdnd Translating mRNA into fertilized eggs of carp or zebra fish, and taking carp or zebra fish juvenile fish with rejected germ cells as a receptor fish for reproductive transplantation of the finless eel;
2) injecting living body fluorescence marker to the finless eel embryo at the 1 cell stage, and selecting the finless eel embryo with the fluorescence marker; selecting embryo tissues behind yolk, digesting the cut eel embryo tissues by using Ringer balanced salt solution containing 0.05-0.3% of collagenase V and 0.5-2% of sodium citrate, lightly blowing the digested embryo tissues by using a 5ml, 10ml or 20ml sterile syringe for 2-10 times at intervals of 10-30 minutes, wherein the total time of enzyme digestion is not more than 2 hours, filtering out undissociated tissue blocks by using a cell sieve with the aperture of 70 mu m or 100 mu m, centrifuging the digested embryo tissues at 2000rpm for 3-8 minutes, re-filtering the re-suspended cells by using a PBS solution containing fetal calf serum and then using a cell sieve with the aperture of 40 mu m or 70 mu m, retaining the filtrate, and observing and confirming whether the filtrate is a single cell suspension with complete appearance under a microscope; performing fluorescence sorting on the single-cell suspension meeting the requirements by using a flow cytometer to obtain GFP positive cells, namely ricefield eel primordial germ cells, injecting the ricefield eel primordial germ cells to gonad primordium positions of carps or zebra fish juvenile fish with endogenous germ cells removed through a glass needle to obtain receptor fish embedded with the ricefield eel germ cells;
3) extracting total RNA of gonads of 1-12 months old recipient carps or zebra fishes after the transplantation of the ricefield eel germ cells, and performing reverse transcription to obtain cDNA;
4) using cDNA obtained in step 3) as template, amplifying ricefield eel germ cell specific expression by nested PCRdazlA gene sequence; the primers of the first round of the nested PCR of PCR are eel-dazl-F1： 5’-AGTATGCCCTCCTTTCTCCT-3’, eel-dazl-R1: 5'-AATTGATGCCTTGATTGTCC-3', respectively; the second round of PCR takes the product of the first round of PCR reaction as a template, and the primer sequences are as follows: eel-dazl-F2： 5’-CAGAGCACCCTCACCATACC-3’, eel-dazl-R2 ：5’- GAATAGCCGAAAGCCCTTAT -3’；
Finless eel obtained by first round PCR amplificationdazlThe fragment is 744bp, and the finless eel obtained by the amplification after the second round of PCRdazlThe fragment is 355 bp; indicating that the integrated ricefield eel germ cells exist in the gonad of the receptor fish.
2. The method according to claim 1, wherein the carp morpholino is: 5'-cctgctgtagctgctgtccctccat-3', respectively;
the morpholino of zebrafish is: 5'-gctgggcatccatgtctccgaccat-3' are provided.
3. The method according to claim 1, wherein the volume ratio of fetal bovine serum to PBS in step 2) in the PBS containing fetal bovine serum is 2%.
4. Use of the method of claim 1 in commercial farming of finless eels.
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| US20080152634A1 (en) * | 2005-01-26 | 2008-06-26 | Herman Pieter Spaink | Means and Methods for Improving the Development and Maturation of Eggs and/or Sperm in Fish Using Hormones Produced by Transplanted Cells |
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| US20080152634A1 (en) * | 2005-01-26 | 2008-06-26 | Herman Pieter Spaink | Means and Methods for Improving the Development and Maturation of Eggs and/or Sperm in Fish Using Hormones Produced by Transplanted Cells |
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