Method for evaluating cold resistance of genetic engineering livestock

Abstract

The invention provides a gene engineering livestock cold resistance evaluation method, and belongs to the technical field of gene engineering animals. According to the method, the cold resistance of the genetically engineered pigs is systematically evaluated from shallow to deep from multiple aspects through the methods of environment low temperature simulation, animal behavior observation, body surface heat radiation detection and transcriptome analysis. The method breaks the cognition of the public that the thicker the backfat of the pig is, the stronger the cold resistance is, and the thinner the backfat is, the weaker the cold resistance is, and the method for evaluating the cold resistance of the genetic engineering pig is obtained, so that the evaluation mode has a more scientific basis.

Description

Technical Field

[0001] The invention relates to the technical field of genetically engineered animals, and in particular to a method for evaluating the cold resistance of genetically engineered livestock. Background Art

[0002] Genetically engineered animals are animals that humans manipulate or modify specific areas of the animal genome based on their subjective will and needs using modern genetics and molecular and cellular bioengineering technologies, thereby completely changing the biological heredity at the genomic level and obtaining animal individuals that can be continuously and stably inherited by future generations. Genetically engineered animals are divided into transgenic animals and gene-edited animals. Among them, animals obtained by introducing exogenous DNA sequences outside the gene pool of the target species into the genome are transgenic animals; animals in which the target site in the genome is precisely deleted or modified to inactivate the target gene or modify the encoded product, and no exogenous DNA sequences outside the gene pool of the target species are introduced are gene-edited animals.

[0003] Genetically engineered animals have significant phenotypes compared to wild-type individuals due to the knockout, modification, editing and insertion of new genes.

[0004] Cold resistance is an important phenotype of livestock. The traditional method of evaluating the cold resistance of pigs is to indirectly evaluate the cold resistance of animals by measuring or detecting the thickness of pig back fat, or to detect cold resistance by cold stimulation.

[0005] For example, in Chinese patent CN107182940A, one-month-old transgenic pigs and wild-type pigs were placed in a 4°C refrigerator for 4 hours. Starting from 0 hours, the rectal temperature of the pigs was measured every hour with an electronic thermometer. Between 1 hour and 4 hours of cold stimulation, the rectal temperature of the transgenic pigs was significantly higher than that of the wild-type pigs. At the same time, the pigs were photographed with an infrared camera to record the changes in the pigs' body surface temperature to detect the piglets' ability to resist cold. However, this method only evaluates the cold resistance of animals by the changes in the pigs' body surface and rectal temperature. For special livestock, it is impossible to judge the cold resistance of animals by a single factor.

[0006] However, with the rapid development of transgenic breeding, the number of genetically engineered livestock species is increasing, and traditional evaluation methods can no longer meet the needs, especially for some special genetically engineered livestock, such as lean transgenic pigs with genes related to energy metabolism.

[0007] For some phenotypes and performance testing and evaluation of genetically engineered livestock, such as cold resistance, if traditional methods such as back fat thickness, behavioral changes, and body surface thermal radiation are used, it is easy to make wrong judgments. The method of evaluating the cold resistance of livestock by testing the back fat thickness of livestock has certain limitations. For example, the back fat thickness can only indirectly indicate the strength of livestock's thermal insulation performance, but cannot indicate the strength of livestock's heat production performance. The strength of livestock's cold resistance is not only to evaluate the thermal insulation performance, but more importantly, the heat production capacity of livestock in low temperature conditions.

[0008] Meishan pigs are a local breed in my country and are famous for their excellent reproductive performance. The highest record is 33 piglets per litter. They are highly adaptable, can tolerate roughage, and have a certain degree of cold resistance. Through research, it has been found that editing the MSTN gene can increase the lean meat rate of pigs, thus making up for the shortcomings of Meishan pig production performance. Myostatin (MSTN) is a classic member of the TGF-β family and was discovered in 1997. MSTN protein is mainly secreted by skeletal muscle and mainly acts on skeletal muscle, playing a key regulatory role in inhibiting the growth and development of skeletal muscle. It can be considered that the secretion of MSTN protein directly maintains the balance between skeletal muscle and fat, and also maintains the balance of energy metabolism. There are natural mutations of MSTN in many species in nature, especially in cattle, sheep and other species, which lead to a large number of MSTN inactivation genotypes and produce double muscle hip phenotypes. So far, in addition to rodent model animal models, the preparation of MSTN gene-edited cattle, goats, sheep and rabbits has been widely reported, and the phenotypes are highly similar to the homozygous double muscle hip phenotypes of natural mutations. However, there is no targeted method to evaluate its cold resistance.

[0009] Therefore, it is necessary to provide a method for evaluating the cold resistance of genetically engineered livestock to reduce judgment errors. Summary of the invention

[0010] In view of this, the present invention provides a method for evaluating the cold resistance of genetically engineered livestock. In addition to traditional methods for studying cold resistance, such as back fat thickness, body temperature changes under cold stimulation, behavioral changes and body surface thermal radiation, the cold resistance of livestock can be directly judged and evaluated by comparing key metabolic genes and energy metabolism methods in the back fat tissue before and after cold stimulation, and the results are more accurate.

[0011] The method for evaluating the cold resistance of genetically engineered livestock of the present invention comprises the following steps:

[0012] (1) Detection of livestock backfat thickness;

[0013] (2) Detect behavioral changes in livestock under low temperature conditions;

[0014] (3) Detect rectal temperature and body surface temperature of livestock in low temperature environment;

[0015] (4) Detect the PDK4 gene expression level in livestock back fat.

[0016] Preferably, the detection of behavioral changes of livestock in a low temperature environment in step (2) is to subject the livestock to a cold exposure treatment at 4°C for 3 hours and then observe their standing movement or curled up state.

[0017] Preferably, the step (3) of detecting the rectal temperature of livestock in a low temperature environment is to subject the livestock to cold exposure at 4°C and then record the rectal temperature every 0.5h.

[0018] Preferably, the detection of the body surface temperature of the livestock in a low temperature environment in step (3) is to subject the livestock to cold exposure treatment at 4°C and then use an infrared thermal imaging system to detect the body surface temperature of the livestock at different times.

[0019] Preferably, the specific method for detecting the PDK4 gene expression level in livestock back fat in step (4) is:

[0020] First, total RNA was extracted from the back fat of livestock, and total RNA reverse transcription and real-time quantitative fluorescence PCR were performed to obtain the expression level of PDK4 gene.

[0021] Generally speaking, in a low-temperature environment, pigs with thick back fat have good thermal insulation performance, while pigs with thin back fat have poor thermal insulation performance. It is generally believed that pigs with thick back fat have stronger cold resistance than pigs with thin back fat. After placing pigs in a low-temperature environment (4 degrees) for 3 hours, their behavioral changes were observed, mainly curling up and standing up. Compared with curling up behavior, standing behavior can increase skeletal muscle movement heat production to maintain body temperature, while curling up behavior can better keep warm.

[0022] With the rapid development of biological breeding technology, traditional evaluation methods are no longer applicable to new animal breeding materials. Therefore, the present invention systematically evaluates the cold resistance of genetically engineered pigs from multiple levels and from the shallow to the deep by simulating low environmental temperature, observing animal behavior, detecting body surface thermal radiation, and analyzing transcriptomes.

[0023] The study found that when the ambient temperature drops, the animal's metabolic mode changes from being dominated by sugar metabolism to being dominated by lipid metabolism. The key gene that regulates this metabolic change is pyruvate dehydrogenase kinase 4 (PDK4). PDK4 gene expression increases when glucose is converted to fatty acids as an energy source.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a method for evaluating the cold resistance of genetically engineered livestock. By simulating low environmental temperature, observing animal behavior, detecting body surface thermal radiation, and analyzing transcriptomes, the cold resistance of genetically engineered pigs is systematically evaluated from multiple levels and from the shallow to the deep. This method breaks the public's perception that the thicker the pig's back fat, the stronger its cold resistance, and the thinner the back fat, the weaker its cold resistance. A method that can be used to evaluate the cold resistance of genetically engineered pigs is obtained, making the evaluation method more scientifically based. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph of back fat thickness of wild-type Meishan pigs and MSTN gene-edited pigs of the same breed;

[0027] Figure 2 This is a diagram of behavioral changes in wild-type Meishan pigs and MSTN gene-edited pigs of the same breed after 3 hours of cold exposure;

[0028] Figure 3 This is a graph showing the changes in rectal temperature of wild-type Meishan pigs and MSTN gene-edited pigs of the same breed after 3 hours of cold exposure;

[0029] Figure 4 This is the infrared radiation diagram of wild-type Meishan pigs and MSTN gene-edited pigs of the same breed after 1 hour of cold exposure;

[0030] Figure 5 This is the infrared radiation diagram of wild-type Meishan pigs and MSTN gene-edited pigs of the same breed after 3 hours of cold exposure;

[0031] Figure 6 The expression levels of PDK4 gene in the back fat of wild-type Meishan pigs and MSTN gene-edited pigs of the same breed. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments.

[0033] Example 1

[0034] A method for evaluating the cold resistance of genetically engineered livestock, comprising the following steps:

[0035] Experimental animals: Wild-type Meishan pigs (WT) and MSTN gene-edited pigs of the same breed (MSTN - / - ), in which the MSTN gene-edited pigs were produced by knocking out the third exon of Meishan pig MSTN, which caused the MSTN gene coding to terminate prematurely and the MSTN protein to be inactivated.

[0036] Experimental method: Three wild-type Meishan pigs (90-day-old boars) and three MSTN - / -Meishan pigs (90-day-old boars), wild-type Meishan pigs and edited Meishan pigs that had been kept at room temperature for a long time were anesthetized before cold exposure, and sodium pentobarbital (brand: MERCK) was used for intravenous injection. After anesthesia, some back fat tissue samples were collected. The operation lasted about 4 hours, and sutures were performed after the collection was completed. After the wound was fully recovered (1 month after surgery), the 6 pigs were placed in cages in a car with a low-temperature control system and subjected to 3 hours of cold exposure at 4°C. After 3 hours of cold exposure, the behavioral changes of the pigs were observed and recorded. At the same time, starting from 0h, the rectal temperature of the 6 pigs was measured every 0.5 hours with an electronic thermometer. After 3 hours of cold exposure, the surface temperature of the pigs was detected using an infrared thermal imaging system (FLIR), and the back fat was immediately collected and the back fat thickness was recorded for slaughter experiments. The experimental results are as follows:

[0037] 1. Collect back fat after slaughter. Figure 1 , Figure 1 The left side of the middle image shows the back fat of MSTN-edited pigs; the right side shows the back fat of wild-type pigs. Figure 1 It can be seen that after 3 h of cold exposure, the slaughter experiment was carried out and MSTN - / - The back fat thickness of pigs was significantly lower than that of wild-type pigs.

[0038] 2. After 3 hours of cold exposure at 4°C, observe behavioral changes. The results are as follows Figure 2 , Figure 2 The left side of the middle image shows an MSTN-edited pig; the right side shows a wild-type pig. Figure 2 It can be seen that MSTN - / - The pigs appear to be in a curled-up position, while the wild-type pigs appear to be in a standing position.

[0039] 3. Starting from 0h, check the rectal temperature of the pig every 0.5h. The results are as follows: Figure 3 , Figure 3 MSTN + / + stands for wild-type pig; MSTN - / - Represents MSTN edited pig; P>0.05.

[0040] from Figure 3 It can be seen that MSTN - / - There was no significant difference in body temperature between the pigs and wild-type pigs. - / - Pig back fat is thinner and has poor thermal insulation performance, MSTN - / - Pigs were more likely to curl up in response to hypothermia, but their core temperatures did not show any significant differences.

[0041] 4. After 1h and 3h of cold exposure at 4℃, the pigs' body surface temperature was detected using an infrared thermal imaging system to generate thermal images. The results are as follows: Figure 4 and Figure 5 , Figure 4MSTN + / + stands for wild-type pig; MSTN - / - On behalf of MSTN editor Pig; Figure 5 The left side of the middle image shows an MSTN-edited pig (lying down), and the right side shows a wild-type pig (standing and moving). Figure 4 and Figure 5 It can be seen that MSTN - / - Pigs have significantly higher thermal radiation than wild-type (WT) pigs, which are close to the ambient temperature ( Figure 4 ). The skeletal muscles of WT pigs showed a higher rate of heat production due to higher locomotion and mastication activities. Therefore, the limbs and face of WT pigs had significantly higher thermal radiation than other parts of the body. In contrast, MSTN - / - The pig lay motionless. Figure 5 ), indicating MSTN - / - Pigs have a greater ability to produce heat.

[0042] 5. Detection of PDK4 gene expression in livestock backfat:

[0043] (1) Prepare tweezers and scissors and bake them in an oven at 180°C for 4 hours. Prepare plastic products (centrifuge tubes, homogenization tubes) using RNase-free products and sterilize them in a high-pressure steam sterilizer before drying to ensure that there is no RNase contamination for total RNA extraction;

[0044] a. Add 1 ml of Trizol cell lysis buffer and homogenization beads to the homogenization tube in advance, mark it and pre-cool it on ice, take about 100 mg of tissue and put it into the pre-cooled homogenization tube, and then homogenize it on a homogenizer;

[0045] b. Place the ground tissue sample in a pre-cooled centrifuge, centrifuge at 4°C, 12,000 rpm for 2 minutes, and transfer the supernatant to a new centrifuge tube;

[0046] c. Add 200 μl of chloroform, shake thoroughly, and place on ice for 20 min; centrifuge at 4°C, 12,000 rpm for 20 min, and aspirate 400 μL of the upper layer into a new centrifuge tube;

[0047] d. Add 400 μL of pre-cooled isopropanol, mix thoroughly by inversion, and place on ice for 10 min. Centrifuge at 4°C, 12,000 rpm for 15 min. At this time, the RNA is centrifuged to the bottom of the tube to form a white precipitate. Discard the supernatant.

[0048] e. Add 1 mL of pre-cooled 75% ethanol, shake and wash the RNA, suspend the RNA in the liquid, centrifuge at 4°C, 12,000 rpm for 5 minutes, and discard the supernatant. Repeat this step once more;

[0049] f. Centrifuge for 30 seconds to transfer the liquid on the tube wall to the bottom of the tube, and aspirate the remaining 75% ethanol with a pipette tip.

[0050] g. Place it in a ventilated area until the RNA at the bottom of the tube is completely dried. Add 10 - 100 μL of enzyme-free and sterile water according to the amount of RNA, and let it stand on ice for sufficient dissolution and mixing to obtain total RNA.

[0051] (2) Total RNA purity detection

[0052] Calibrate and zero the differential spectrophotometer. Take 1 μL of total RNA and measure OD230, OD260, and OD280 respectively, and calculate OD260 / OD280 and OD260 / OD230. The normal range should be 1.8 < OD260 / OD280 < 2.0. If it is not within this range, RNA needs to be extracted again; the remaining samples are stored at -80 °C for future use.

[0053] (3) Total RNA reverse transcription

[0054] The total RNA reverse transcription reaction system is shown in Table 1:

[0055] Table 1

[0056]

[0057]

[0058] Put the total RNA into a PCR instrument, and set the program: the first step is 42 °C for 2 minutes; the second step is 37 °C for 15 minutes; 85 °C for 5 seconds; 4 °C for ∞ to terminate the reaction, and finally store it in a -20 °C refrigerator.

[0059] (4) Real-Time quantitative PCR (RT-qPCR)

[0060] Use Primer Primer5 software to design primers for the target gene and the internal reference gene 18S, and detect the primer specificity by NCBI Primer-BLAST. They are synthesized by Beijing Tsingke Biotechnology Co., Ltd. The relevant primer sequences are shown in Table 2:

[0061] Table 2

[0062]

[0063] The RT-qPCR reaction is carried out according to the instructions of the TB GreenTM Premix Ex TaqTM (TaKaRa, RR420A) kit and placed in an ABI7500 instrument for PCR amplification. Each sample is set with 3 technical replicates, with 18S as the internal reference. The reaction system is shown in Table 3:

[0064] Table 3

[0065]

[0066]

[0067] Set the reaction program: pre-denaturation at 95°C for 34 seconds; denaturation at 95°C for 5 seconds, 60°C for 34 seconds, 40 cycles; 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds, 1 cycle.

[0068] The wild-type pig gene expression at room temperature was used as standard 1, the wild-type pig cold exposure state, MSTN - / - Edit Pig Room Temperature and MSTN - / - The gene expression levels of pigs under cold exposure were compared with those of pigs, and the results of the backfat PDK4 gene expression levels were obtained as follows: Figure 6 .from Figure 6 It can be seen that the expression level of PDK4 gene in the back fat of wild-type pigs after cold exposure is 137 times that of wild-type pigs at room temperature (average ratio); MSTN - / - The expression level of PDK4 gene in backfat of edited pigs after cold exposure is MSTN - / - Edited pigs at room temperature 151 times (average ratio). This means that under low temperature conditions, MSTN - / - The intensity of lipid metabolism in edited pigs is higher than that in wild-type pigs, further proving that MSTN - / - Pigs have a greater ability to produce heat.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for evaluating the cold resistance of genetically engineered livestock, characterized in that: The following steps are involved: (1) Detection of livestock backfat thickness; (2) Detect behavioral changes in livestock under low temperature conditions; (3) Detect rectal temperature and body surface temperature of livestock in low temperature environment; (4) Detect the PDK4 gene expression level in livestock back fat.

2. The method for evaluating cold resistance of genetically engineered livestock according to claim 1, characterized in that: The detection of behavioral changes of livestock in a low temperature environment in step (2) is to subject the livestock to a cold exposure treatment at 4° C. for 3 hours and then observe their standing movement or curled-up state.

3. The method for evaluating cold resistance of genetically engineered livestock according to claim 1, characterized in that: The step (3) of detecting the rectal temperature of livestock in a low temperature environment is to subject the livestock to cold exposure at 4°C and then record the rectal temperature every 0.5h.

4. The method for evaluating cold resistance of genetically engineered livestock according to claim 1, characterized in that: The step (3) of detecting the body surface temperature of the livestock in a low temperature environment is to subject the livestock to cold exposure treatment at 4°C and then use an infrared thermal imaging system to detect the body surface temperature of the livestock at different times.

5. The method for evaluating cold resistance of genetically engineered livestock according to claim 1, characterized in that: The specific method for detecting the PDK4 gene expression level in livestock back fat in step (4) is: First, total RNA was extracted from the back fat of livestock, and total RNA reverse transcription and real-time quantitative fluorescence PCR were performed to obtain the expression level of PDK4 gene.

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