阅读说明：本技术 一种提高转基因效率的鸡胚背主动脉注射方法 (Chick embryo dorsal aorta injection method for improving transgenic efficiency ) 是由 郑喜邦 李恭贺 李非 韦金鱼 韦茏芹 徐小明 邢青波 刘灵康 韦宗海 苏立燧 唐 于 2020-03-19 设计创作，主要内容包括：本发明公开了一种提高转基因效率的鸡胚背主动脉注射方法,包括对发育2.5天的鸡胚进行壳外背主动脉注射腺相关病毒颗粒,注射后的鸡胚转入受体蛋(双黄蛋)壳中继续孵化出壳。本发明通过改良的背主动脉注射方法实现了对鸡胚背主动脉的壳外注射,提高了注射的准确率,也实现了对鸡胚发育过程的直视观察,其胚存活率、孵化率和转基因表达效率明显高于胚盘下腔注射组和传统背主动脉注射组,提高了转基因鸡胚孵化率和EGFP阳性检出率。(The invention discloses a chicken embryo dorsal aorta injection method for improving transgenic efficiency, which comprises the steps of injecting adeno-associated virus particles into the dorsal aorta of a chicken embryo which grows for 2.5 days, transferring the injected chicken embryo into a recipient egg (double-yolk egg) shell, and continuously hatching the egg shell. According to the invention, the extracapsid injection of the chick embryo dorsal aorta is realized through the improved dorsal aorta injection method, the injection accuracy is improved, the direct-view observation of the development process of the chick embryo is also realized, the embryo survival rate, the hatching rate and the transgene expression efficiency are obviously higher than those of a blastoderm inferior cavity injection group and a traditional dorsal aorta injection group, and the transgenic chick embryo hatching rate and the EGFP positive detection rate are improved.)

1. A chicken embryo dorsal aorta injection method for improving transgenic efficiency is characterized by comprising the following steps:

a, placing a newly-fertilized egg in an incubator, and culturing for 2.5 days at the temperature of 37.8 ℃ and the relative humidity of 60%;

b, slowly pouring the whole chick embryo of the fertilized egg into a weighing plate;

c, injecting virus particles into the chicken embryo through the dorsal aorta;

d, selecting the double-yolk egg as the receptor egg, sterilizing the receptor egg before windowing, and discarding the content after windowing the blunt end;

E. transferring the injected whole chick embryo into the egg shell of the receptor, sealing the window of the egg shell of the receptor by using a preservative film, enabling the window to be upward, and putting the egg shell into an incubator to continue culturing until the egg shell is removed.

2. The method for injecting the aorta dorsum of chicken embryo for improving transgenic efficiency as claimed in claim 1, wherein the sterilization method comprises: the outer surfaces of the fertilized eggs and the eggshells of the recipients are cleaned by 0.01 percent benzalkonium bromide solution and then are sprayed with 75 percent alcohol for disinfection.

3. The method for injecting the aorta dorsum of chick embryo for improving transgene efficiency as claimed in claim 1, wherein said windowing of the recipient egg comprises windowing at the blunt end of the recipient egg shell with a diameter of 40 mm.

4. The method for injecting the chick embryo dorsal aorta with the improved transgenic efficiency as claimed in claim 1, wherein the method for injecting the chick embryo dorsal aorta comprises injecting 2 μ l of virus particles into the chick embryo dorsal aorta through a micro injection needle under a stereoscopic microscope.

5. The method for injecting transgenic chicken embryo dorsal aorta with improved transgenic efficiency as claimed in claim 1, wherein the sealing film is Handi-Wrap preservative film.

6. The method of claim 1, wherein the fertilized egg and the recipient egg are from the same species.

7. The method of claim 1, wherein the double yolk eggs are used as the receptor and weigh 30-40g more than the corresponding fertilized eggs.

Technical Field

The invention relates to the technical field of transgenic biology, in particular to a chick embryo dorsal aorta injection method for improving transgenic efficiency.

Background

Since the 80's of the 20 th century, avian transgenesis has been one of the hot spots of research in the biotechnology field. The poultry transgenosis has important application value in the aspects of improving the laying rate of poultry meat production, improving disease resistance, establishing a bioreactor model and the like. Compared with a mammal bioreactor, the poultry oviduct bioreactor has the advantages of short incubation time (average 3 weeks), short generation interval (average 20 weeks), high propagation speed, low production cost, high yield and the like.

Transgenic chicken production is technically challenging because the chicken eggs are tightly connected to the yolk and cannot be cultured in vitro as mammals do to produce transgenic chickens by nuclear transfer techniques. Until 1988, Perry developed a chicken embryo culture System (System I-III). Currently, the shell-changing culture method of System II and System III is commonly used. In System II stage, the donor whole embryo from the freshly fertilized egg is transferred to the eggshell of a recipient (weighing about 3-5g more than the donor); in System III stage, chick embryos cultured for 2.5 days are transferred to the eggshells of larger recipients (30-40 g heavier than the donor) and culture is continued until the eggshells emerge. Although the subgerminal cavity injection method is simple in technology, the injected chick embryos need to be replaced twice, mechanical damage is caused to the chick embryos, and the survival rate of the chick embryos is low.

The traditional dorsal aorta injection method is to open a window through the blunt end of an eggshell, inject exogenous genes into the dorsal aorta of an HH14-16 stage chick embryo, close the window, and continuously hatch and shell. The traditional dorsal aorta injection method has the advantages that shell replacement culture is not needed, and the defects that the injection precision in the shell is low, the transgenic efficiency is reduced, and the development process of transgenic chick embryos cannot be observed directly. Therefore, the conventional dorsal aortic injection method is to be improved.

Disclosure of Invention

The invention aims to provide a chick embryo dorsal aorta injection method for improving the transgenic efficiency in order to solve the existing problems.

In order to achieve the purpose, the invention adopts the following technical scheme:

a chicken embryo dorsal aorta injection method for improving transgenic efficiency comprises the following steps:

a, placing a newly-fertilized egg in an incubator, and culturing for 2.5 days at the temperature of 37.8 ℃ and the relative humidity of 60%;

b, slowly pouring the whole chick embryo of the fertilized egg into a weighing plate;

c, injecting virus particles into the chicken embryo through the dorsal aorta;

d, selecting the double-yolk egg as the receptor egg, sterilizing the receptor egg before windowing, and discarding the content after windowing the blunt end;

E. transferring the injected whole chick embryo into the egg shell of the receptor, sealing the window of the egg shell of the receptor by using a preservative film, enabling the window to be upward, and putting the egg shell into an incubator to continue culturing until the egg shell is removed.

Specifically, the sterilization method comprises; the outer surfaces of the fertilized eggs and the eggshells of the recipients are cleaned by 0.01 percent benzalkonium bromide solution and then are sprayed with 75 percent alcohol for disinfection.

Further, the method for windowing the acceptor egg comprises the step of windowing the blunt end of the acceptor egg shell, wherein the diameter of the window is 40 mm.

Specifically, the method for dorsal aortic injection comprises injecting 2 μ Ι of viral particles into the chick embryo dorsal aorta under a stereomicroscope through a microscopic injection needle.

Furthermore, the sealing film adopts a Handi-Wrap preservative film.

In particular, the fertilized egg and the recipient egg are both from the same species.

Furthermore, double yellow eggs are selected as receptors, and the weight of the double yellow eggs is 30-40g than that of the corresponding fertilized eggs.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, the ex-shell injection of the chick embryo is realized through the improved back aorta injection method, the injection accuracy is improved, the chick embryo after injection is transferred into a substitute egg shell (double-yolk egg) to be cultured until the chick embryo hatches out of the shell, and the direct-view observation of the development process of the chick embryo is also realized, the embryo survival rate, the hatching rate and the transgenic expression efficiency are obviously higher than those of a blastoderm lower cavity injection group and a traditional back aorta injection group, and the transgenic chick embryo hatching rate and the EGFP positive detection rate are improved.

Drawings

FIG. 1 is a diagram of the improved dorsal aortic injection and remodeling procedure set forth in the present invention;

FIG. 2 is a bar graph illustrating the effect of different injection methods on chick embryo survival according to the present invention;

FIG. 3 is a bar graph illustrating the effect of different injection methods on chick embryo hatchability according to the present invention;

FIG. 4 is a bar graph of the effect of different injection methods on the EGFP expression efficiency of chicken embryos according to the present invention;

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Fertilized eggs and non-fertilized eggs (system II standby receptors) used in the test are purchased from Guangxi Nanning Rich phoenix farming-grazing GmbH, double yellow eggs (system III standby receptors) are purchased from Nanning Liangfeng farming-grazing GmbH, and AAV particles for injection (HBAAV-CMV-EGFP) are purchased from Shanghai Han Heng Biotechnology GmbH, and the titer is 1X10^8 TU/ML.

The experimental groups are respectively a hypocotyl hypo-cavity injection group of chicken embryos in stage X (hereinafter referred to as a hypocotyl hypo-cavity injection group), a dorsal aorta injection group of chicken embryos in stage HH14-16 (hereinafter referred to as a traditional dorsal aorta injection group), and an improved dorsal aorta injection group of chicken embryos in stage HH14-16 (hereinafter referred to as an improved dorsal aorta injection group). Each test group was injected with 30 fertilized eggs at a time, and 5 independent tests were performed, each group being injected with 150 fertilized eggs. The blank control group was not treated, and 12 fertilized eggs were incubated each time, and 60 fertilized eggs were incubated in total.

And during the hatching period of the chick embryos, observing the development condition of the chick embryos every day, and recording the survival rate of the chick embryos. And after the chick embryos are hatched and shelled, counting the hatching rate. And (3) irradiating the fresh dead chicks or the chicks with a fluorescent protein flashlight to observe whether green fluorescent protein (EGFP) is expressed on the body surfaces and the visceral tissues of the chicks, and counting the positive rate of the EGFP.

Subgerminal cavity injection method: adopting newly-fertilized eggs as donors, selecting non-fertilized eggs which are 3-5g heavier than the donors as System II acceptors, weighing, pairing one by one, cleaning the fertilized eggs and the outer surfaces of the eggshells of the acceptors by 0.01 percent benzalkonium bromide solution, and spraying 75 percent alcohol for disinfection. A window with a diameter of 32mm was cut at the tip of the recipient egg with a micro electric grinder, the contents were discarded, and the inner and outer surfaces were rinsed with distilled water. The recipient eggs were placed window down in a ceramic dish lined with moist gauze. Lightly breaking fertilized eggshells in an ultraclean workbench, transferring the whole embryo into a SystemII receptor eggshell to enable an embryonic disc to float upwards, connecting a mouth suction tube with a microinjection needle, irradiating by virtue of a cold light source under a stereomicroscope, injecting 2 mul of EGFP-AAV virus into the lower cavity of the embryonic disc, sealing an eggshell window by a SaranWrap preservative film (5cm multiplied by 5cm), placing the eggshell window downwards into a chick embryo incubator, and culturing for 2.5 days under the conditions that the temperature is 37.8 ℃ and the relative humidity is 60%. For surviving chick embryos, 30-40g of double yolk eggs were selected as System iii receptors compared to the corresponding donor weight. The processing method of the acceptor eggshell is the same as that of System II, except that a window is opened at the blunt end of the acceptor eggshell, and the diameter is 40 mm. Transferring the surviving chick embryos to an eggshell of a System III receptor, sealing a window with an upward Handi-Wrap preservative film, putting the chick embryos into an incubator, and continuously culturing until the chick embryos are hatched.

Traditional dorsal aortic injection: and (3) placing the newly-laid fertilized eggs in an incubator for incubation, and after incubating for about 2.5 days, keeping the blunt ends of the fertilized eggs upward and horizontally standing for 1-2 hours. The air chamber position was determined using an egg candler and marked with a pencil. Wiping an eggshell with 75% alcohol cotton, opening a window with the diameter of 1-1.5cm in the center of a pencil marking area by using a forceps, dripping a little sterilized normal saline on the surface of a shell membrane, tearing the shell membrane, connecting a mouth suction tube with a micro-injection needle, and injecting 2 mu l of EGFP-AAV virus liquid into a chick embryo back aorta. The window was closed with a sealing film (Parafilm). And (5) keeping culturing until the young chicken is hatched with the upward window.

FIG. 1 shows a process diagram of an improved dorsal aortic injection and remodeling procedure; modified dorsal aortic injection method freshly fertilized eggs were used as donors, weighed and recorded, and placed in an incubator set at 37.8 ℃ and 60% relative humidity. For chick embryos that have developed to the HH14-16 stage (about 2.5 days), double yolk eggs weighing 30-40g of the corresponding donor were selected as recipients. The method of treatment of the recipient eggshells is as same as the subgerminal cavity injection method System III. Wiping the surface of the donor eggshell with 75% alcohol, slightly breaking the eggshell in an ultra-clean bench, pouring the whole embryo into a disposable weighing tray, enabling the chick embryo to face upwards, connecting a mouth suction tube with a micro-injection needle, and injecting 2 mu l of EGFP-AAV virus into the chick embryo dorsal aorta under a stereoscopic microscope. And (3) moving the injected chick embryos into the eggshells of the receptors, sealing the window by using a Handi-Wrap preservative film, placing the chick embryos into an incubator with the window facing upwards, and continuously culturing until the chick embryos are hatched.

The survival rates of transgenic chick embryos cultured to 8, 14, 18 and 21 days and the EGFP positive detection rate after 14 days to shelling were calculated. The SPSS22.0 software single-factor analysis of variance method is adopted to analyze the difference significance among different experimental groups. P <0.05, indicating significant difference; p <0.01, indicating that the difference is very significant.

Results

As shown in FIG. 2 and Table 1, the survival rate of chick embryos was not significantly different between the chick embryo development day 8 and the chick embryo survival rate in the blastoderm cavity injection group and the conventional dorsal aortic injection group (P > 0.05). However, the survival rate of chick embryos in the modified dorsal aorta injection group is higher than that in the traditional dorsal aorta injection group, and the difference is remarkable (P < 0.01). Over time, chick embryo survival rates generally show a downward trend. However, when chicken embryos developed to days 14, 18 and 21, the survival rate was significantly higher in the modified dorsal aortic injection group than in the subgerminal injection group (P <0.05 or P <0.01) and the conventional dorsal aortic injection group (P < 0.01).

TABLE 1 Effect of three injection methods on chick embryo survival

As shown in fig. 3 and table 2, the hatchability of transgenic chick embryos in the modified dorsal aortic injection group (37%) was significantly higher than that in the subgerminal cavity injection group (26%) and that in the conventional dorsal aortic injection group (16%) (P < 0.01).

TABLE 2 Effect of three injection methods on chick embryo hatchability

Observing the expression condition of green fluorescent protein by using a fluorescent protein flashlight and an adaptive filter lens of a fresh dead chick or a chick with a neck, wherein the result shows that EGFP is expressed in the heart, gall bladder, sternum, skin, beak and other parts; the GFP positive rate of chick embryos in the modified dorsal aortic injection group is obviously higher than that in the subgerminal cavity injection group and the traditional dorsal aortic injection group (figure 4, table 3) (P < 0.01).

TABLE 3 Effect of three injection methods on chick embryo EGFP expression efficiency

In this test, one of the causes of the gradual decrease in the survival rate of the embryo from System II to System III may be the damage to the embryo due to the embryo shelling operation. In addition, subgerminal injection and dorsal aortic injection also cause mechanical damage to the embryo, which is also responsible for embryo death. The hatchability of the transgenic chick embryos in the improved dorsal aorta injection group in the test is higher than that of the traditional dorsal aorta injection group because the former only needs 1 shell replacement culture operation and the mechanical damage to the chick embryos is smaller than that of the latter.

Microinjection at different developmental stages may directly affect the survival rate of subsequent embryos and the expression efficiency of foreign genes. Primordial Germ Cells (PGCs) are considered to be ideal target cells for the production of transgenic chickens as sperm and egg precursor cells, and it is desirable to obtain transgenic chickens by in vivo transfection or transduction of PGCs. The newly fertilized egg is already in the HH 10 stage of the development of the chicken embryo, 5 to 6 ten thousand embryonic cells exist in the lower cavity of the blastoderm between the blastoderm and the yolk, but only 40 PGCs exist; in the HH14-16 phase (about 2.5 days old), PGCs enter the blood circulation as blood islands and vessels form, increasing in number to 100-200. This is also why the EGFP positive rate of the transgenic chicken embryos in the modified dorsal aorta injection group of this experiment was higher than that in the subgerminal injection group. Compared with the traditional dorsal aorta injection method, the improved dorsal aorta injection method realizes extracapsid dorsal aorta injection, improves the injection accuracy and further improves the EGFP positive rate of the embryo tissues of the transgenic chickens.

The distance of ethnic relationships (interval between ethnicities) is also another factor that affects hatchability. The separation of diluted egg white from chicken, turkey, guinea fowl and duck eggs as the acceptor egg white and egg shells as the System stage iii acceptor, and the hatchability of the chicken embryos in these different acceptor egg whites and the same acceptor egg shells was 60.4%, 55.3%, 47.9% and 19.1%, respectively, which demonstrated the highest hatchability of the chicken embryos in egg white and egg shells. Therefore, the death rate of the chick embryo can be reduced by selecting the poultry eggs with closer relativity as receptors to carry out shell replacement culture. Egg white and egg shells are used as receptors in the stages from System II to System III of the test, so that the risks are avoided, and the death rate of the chick embryos is reduced to a certain extent.

Avian embryos are highly dependent on eggshells during their development, which not only protect the embryos from physical stress and microbial infection, but also regulate moisture and provide minerals. The thickness of the eggshell of the receptor and the material of the sealing film influence the hatching rate. Improving oxygen supply through the recipient eggshells is an important factor in embryo in vitro culture, and different recipient eggshells can cause different hatchability rates. In the experiment, the improved dorsal aorta injection group and the blastoderm inferior cavity injection group both adopt the double-yolk egg as a System III receptor, and despite the mechanical damage caused by microinjection operation, the hatchability of the transgenic chick embryos of the improved dorsal aorta injection group and the blastoderm inferior cavity injection group respectively reaches 37 percent and 26 percent. At System III, when the receptor window was closed, the hatchability after Handi-Wrap membrane sealing was improved by 30% compared to Saran Wrap membrane.

In conclusion, the improved dorsal aorta injection method realizes the extracapsid dorsal aorta injection of the chick embryos and the direct-view observation of the development process of the chick embryos, improves the injection accuracy, and further improves the hatching rate and the transgenic expression efficiency of the transgenic chick embryos.

According to the experiment, the injection of the dorsal aorta outside the shell and the shell replacement culture of the System III are combined, the traditional dorsal aorta injection method is improved, the injection accuracy is improved, and the survival rate, the hatching rate and the transgenic expression efficiency of transgenic chick embryos are improved.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.