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Mutant

Permanent changes in nucleosides at specific sites in the nucleotide chain

Mutation refers to the permanent change of one or more nucleosides at a specific location of the nucleotide chain. The mutant strain is called mutant strain. Mutation may involve the replacement, deletion or rearrangement of one or more nucleoside pairs on the chromosome. This mutation is called point mutation. The wrong aggregation of DNA may increase the mutation rate. Usually the gene mutates at any point probability It is very low (10-5-10/generation). Moreover, in most cases, mutations are harmful, but specific mutations often make microorganisms better adapt to the environment and improve their own Biocatalysis Performance. Therefore, it is difficult to isolate those mutants with excellent characteristics.

Chinese name: Mutant
Foreign name: mutant

Definition: One or more occurrences mutation
Carrier: Biological population 、 gene or chromosome

catalog

▪ Retinal degeneration mutant mice
▪ Desalination of lethal gene mutant mice
▪ Needle tail mutant mice
▪ Oligodactyly mutant mice
▪ Hypertensive mutant rats
▪ Epileptic mutant rats

definition

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mutant

Due to the occurrence of one or more mutation But different from the corresponding wild type Biology 、 Biological population 、 gene or chromosome 。

application

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microorganism Mutants in genetics Research, genetic engineering technology and breeding It has a wide range of applications.

type

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Its types include: (1) Morphological mutant , such as impact Cellular structure 、 Cell morphology 、 Colony morphology and Plaque morphology Mutant strains of; (2) Physiological and biochemical mutant , such as nutritional deficiency mutants and effects sugar Decomposition and utilization of or pigment Production of mutant strains; (3) Resistant mutant , such as Drug resistance 、 Bacteriophage resistance and UV resistance Isomutant. (4) Conditional lethal mutant , such as Temperature sensitive mutant Etc.

Gene mutation

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Fixed-point mutation

mutation , on biology The meaning above refers to the genetic genes (Generally refers to DNA or RNA For animals, including the nucleus and mitochondrion Plants also include chloroplast A permanent, heritable change.

Gene mutation can be divided into spontaneous and induced. The reason may be that the replication of genetic genes is wrong during cell division, or chemical substances 、 radial , or Viruses Impact. Generally, it has four characteristics: 1, recurrence and reversibility of mutation; 2. Mutation multidirectional and multiple alleles. 3. Harmfulness and advantage of mutation; 4. Parallelism of mutation.

Mutation usually leads to abnormal cell operation or cell death, even in higher organisms cancer 。 But at the same time, mutation is also regarded as the driving force of species evolution: undesirable mutations will be eliminated through natural selection, while favorable mutations will be accumulated. Neutral mutations have no effect on species and gradually accumulate, leading to discontinuous balance.

Density mutant

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Denominator Virus particle Contained in nucleic acid The increase or decrease of the amount causes the floating of virus particles density Mutants that have changed. λ. T7 and other phage DNA are partially deleted and become shorter. If the missing part does not contain essential information, infection particles can also be formed. Others have larger molecular weight than wild type due to integration of DNA fragments of host bacteria. Although these phage particles DNA Different content, but shell protein Their density is different from that of wild type bacteriophages. Various particles with different DNA content can be separated by density gradient centrifugation such as cesium chloride.

Screening of yeast mutants

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Materials and methods

1.1 Materials

Screening mutant

1.1 Strains and Plasmids Escherichia coli strain DH5 α is stored in this room; Yeast strain INVSc1 purchased from Invitrogen company Genotype: MET/METahis3 △ 1/his3 △ 1leu2/leu2trp1-289/trp1-289ura3-52/ura3-52Phenotype: His, Leu, Trp, Ura.pHSS6 Yeast Escherichia coli shuttle plasmid (The inserted fragment of pHSS6 contains mTn-3xHA/lacZ homologous recombinant) U.S.A Courtesy of Professor Snyder M of Yale University.

1.2 Methods

1.2.1 50 μ g/mL for planking Kanamycin Or/and 3 μ g/mL tetracycline The resistant clones were screened at 37 ℃ for 12~20h. The clone was washed with LB, properly diluted, and partially frozen with glycerol. After proper dilution, it was inoculated with 50mLLB-50 μ g/mL kanamycin and/or 3 μ g/mL tetracycline, and cultured at 37 ℃ for 6h to exponential growth period.

1.2.2 Extraction, purification and detection of plasmids The conventional methods include extracting or extracting plasmids in large quantities, purifying plasmids, electrophoresis analysis, and detection with ultraviolet spectrophotometer OD260/280. Partial alcohol precipitate preservation.

1.2.3 yeast 3 μ g purified plasmid was transformed and digested by NotI endonuclease (according to the instructions attached to endonuclease), electrophoresis 10 mLINVSc1 yeast was cultured to exponential growth period (30 ℃, 36~48h), OD600=1, centrifuged to collect cells, and washed once with 5mL transformation buffer (transformation buffer: 0.2mol/LLiAC, 40% PEG4000100 μ mol/L β - ME (mercaptoethanol)). The cells were resuspended in 1mL transformation buffer solution, containing 1mg of denatured salmon sperm DNA. After mixing, 100 μ L/tubes were divided into new Eppendorf tubes. Each tube was added with 0.5 μ g of plasmid DNA digested with NotI. After thorough mixing, the cells were incubated at 45 ℃ for 30min. Cells were collected by centrifugation, suspended in 400 μ L SC ura, and laid with 200 μ L boards at 30 ℃ for 2 days.

1.2.4 The expression of LacZ gene is to detect the expression of LacZ gene, select the clones, and lay them in YPAD plates. The density of each plate is 100 pieces. Use sterile filter paper to copy the above plate to another SC ura Flat Above 37 ℃ overnight. Take out the filter paper, with the clone side facing up, and place it in a closed container. Add 10mL chloroform to the bottom of the container for 10~30min. Take out the filter paper, with the clone side facing up, and place it on the X-gal plate (120g X-gal, 0.1mol/L NaPO4 (pH7), 1mmol/LMgSO4, in the 1.6% agar plate), 30 ℃, 2d. Find X-gal positive clones, re select monoclonal clones from YPAD plate and put them on SC ura plate for scribing culture. Freeze the expanded yeast strain (- 80 ℃). The strain with the strongest LacZ expression was selected for large-scale culture.

result

Gene mutation

2.1 Growth of transformants on SC ura medium Since the transformants after homologous recombination contain tetr resistance gene and yeast auxotroph selection marker Ura3, we can select the required transformants through SC ura or tetr.

2.2 LacZ gene expression

If the inserted fragment matches the yeast genome reading frame, the β encoded by LacZ gene- Galactose The N-terminal of glycosidase binds to the C-terminal encoded by the host to produce biological activity It reacts with X-gal to form blue.

2.3 Electrophoresis after digestion

After the purified plasmid is digested by NotI enzyme, a relatively uniform band of about 2.1kb (vector pHSS6) and a relatively uneven band of DNA insertion fragments (about 8kb, containing mTn-3xHA/LacZ) should be visible.

Example - Mutant strain

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Muscular atrophy mice

Dystrophia Muscularis (dy for short) or muscular dystrophy, or hindlimb paralysis mice

The mouse has similar muscular atrophy to human symptom , whose dy/dy is about born fortnight It can be seen that the hind limbs are dragging the ground, showing progressive muscular weakness and extensive muscular atrophy. The diseased mice are alive at birth, but only a few of them can survive for more than 10 weeks reproduction power. However, it has been proved that the ovaries of dy/dy female mice transplanted into normal female mice are fertile. Because the female parent is basically sterile. Due to impotence in mating and poor maternal nature, it is generally heterozygous. If male mice have difficulty in mating with normal female parents, it can be artificially fertilized or ovary Migration. REJ129/dy hind limb hemiplegic mice are commonly used.

Obese mice

(Obese, ob for short)

Experimental mice

Obese mice (ObeseMice) and obese rats (ObeseRat) are obese rats. Obese mice can weigh up to 60g, and this kind of mice has no fertility , manifested as simple obesity without accompanying diabetes 。 Homozygote It can be recognized at about four weeks of age. At this time, its weight gain accelerates, and soon reaches three times the weight of normal littermates. Moderate overeating can make them almost inactive, but when they are young blood sugar And immune activity insulin There is no significant increase. After 5-6 months of age, obesity tends to be stable. Insulin and glucose These mice are not affected by foreign insulin when their insulin levels rise, but food restriction can increase their sensitivity to insulin and prolong their life 。 All female mice were sterile. Ovary and uterus atrophy, if adhere to a moderate diet, male rats can occasionally reproduce. The proliferation of Langerhans Island is related to the increase of insulin secretion, glomerulus Renal nodular fat hyaline lesion occurred, which was limited to the inner surface of the glomerular membrane and basement membrane under the electron microscope. Subcutaneous, retroperitoneal and gonad This is the result of the increase in the number and volume of fat cells, so it is called hypertrophic - proliferative obesity.

The obesity of this kind of mice is very similar to that of human beings. This kind of mouse has been used to carry out many biochemical Pathology , hormone and drug therapy. Because this kind of mouse has no fertility, it must be used Heterozygote Mate to keep this gene 。 Ob/+and+/+are difficult to distinguish. They behave exactly the same. It takes a long time to mate with them time Can be identified. The maintenance of this mutant mouse is very difficult. If diet control leads to weight loss, the effect of maintaining the strain is not good. You can also take eggs from the ovaries of obese mice and transplant them to+/+mice for reproduction. In addition to the ob mutation, ad was later found to be adult obesity and diabetes. The body weight of ad/ad type obesity is twice that of normal mice, which is the same as that of ob/ob type obesity. It is usually sterile and appears at 7-10 weeks of age hyperglycemia And diabetes. stay New Zealand There is also an obesity called NZO (New Zealand and Obese) in mice. This mouse usually has reproductive capacity. All mice are obese, but fat deposition is mainly in the abdomen. Fat deposition can be found at the age of 2 to 4 months, and eventually reaches 70% of the total body weight. Hyperglycemia is not obvious, but the level of insulin increases. During rapid weight gain, food The intake increased significantly. In addition, there is another kind of fat, the gene symbol is fa, obesity also has diabetes, and the pregnancy rate is low. Japan KK mice are also obese. It is known from the obesity of mice that obesity has different genetic types. This is very helpful for the study of a series of problems of human obesity.

Dwarf rat

Dwarf Mice (dw for short)

This deformity is also called Pituitary dwarfism (PituiaryDwarfism), because it lacks brain Hypophysis Anterior lobar auxin and Thyrotropin Therefore, the growth and development are impeded. Homozygous dw/dw mice can be recognized at the age of 12 to 13 months. They are characterized by short tail and short nose. At the mature age, their volume is about 1/4 of that of normal mice in the same nest. At the age of 8 weeks, their weight is 8 to 10 grams. Although most of them can survive to the mature stage, neither male nor female mice have fertility, and secondary myxedema can occur. Feed sick mice the pituitary The fragment of anterior lobe can make the growth rate basically normal. Because the mouse has no fertility and is sterile in both sexes, only heterozygotes can be used to retain the gene, so that only 25% of the dwarf and deformed mice can be obtained. At the age of 9 months, normal pituitary gland can be transplanted into the kidney of this mouse, and the male can grow up to be close to the normal mouse body and reproduce (but no effect can be obtained in female mice, the reason is unknown). This mutant mouse is of great use in endocrine research, such as the analysis and research of "auxin".

Diabetic mice

(Deabytes, db for short)

Hummel et al. (1966) first reported that it was caused by a single recessive mutant gene, which was spontaneously generated from inbred mice (C57BL/Ks) in Jackson's laboratory. This gene (db) and obesity (ob) are not Allele Although their phenotypic characteristics are similar, the first expression of db/db gene mice is that when the diseased mice reach 3-4 weeks of age Fat The blood glucose increased from the normal level below 200mg/100ml blood to 682mg/100ml blood at the age of one year (the average is 563.2mg/100ml blood). Female mice have no fertility, but their ovaries can still restore reproductive activity after being transplanted to other mice. Most mice can not exist for more than 8 months, but if they are fed at an early stage of the disease, their life can be prolonged. Clinical symptoms include obesity, hyperglycemia, diabetes, proteinuria, thirst, polyuria, and finally death due to ketonuria. Postmortem changes include a small number of beta particles in islet cells and pancreatic duct swelling. Like (1972) described the changes in the glomeruli of db/db mice, which are the same in appearance as those of normal mice of the same age, but much larger than the latter diabetes At the age of onset, the changes of glomerular membrane are more obvious, the peripheral basic layer continues to thicken, and there are many summaries. It is also believed that polyuria may be hyperemia sugar The result of diuretic effect on glomerulus.

Skeletal Sclerosis Rats

(Ostepetrosis, op for short)

Experimental mice

It occurs in dwarf mice. This recessive gene (op) is located on chromosome 12. At about 10 days of age (op/op), it can be recognized that its head is dome shaped, its feet are short, and its incisors are absent. The main bone defects are caused by poor bone reconstruction. It is most obvious 6 weeks before and after birth. With the growth of mice, bone formation slows down, and the speed of bone elimination and formation during reconstruction is almost the same. Therefore, the symptoms are relatively minor. The osteoclasts of diseased mice are smaller than those of normal mice in the same litter, Acid phosphatase appears in the whole cell, not only in the interface between osteoclast and bone. The long bones of op/op mice are most affected. At least in the first 6 months of life, the bone marrow cavity is closed and the original sponge bone appears. The bone slices of the diseased mice seem to be denser than those of the normal mice in the same litter. When the diseased mice reach the age of 6 to 10 months, the bone marrow cavity reappears and is full of normal hematopoietic components Megakaryocyte The number of has increased. Unlike other osteosclerotic mutants, they do not accompany Abnormal pigment , so it is different from microphthalmia mi/mi; And Grey Lethal gl/gl mice. The op/op mutant survived for a long time, and the bone lesions disappeared with the growth of mice. Before the bone lesions were eliminated, the ratio of bone matrix formation and thyroid parafollicular cell level significantly decreased.

Due to abnormal bone growth of the diseased mice, Bone Hard, bone marrow cavity disappears, all bones are sclerotic, and accompanied by edentulous. Therefore, dead offspring are often found in the same litter. In the second nest, the mice that died after weaning had no teeth and died because they could not eat, but they could be kept by feeding soft feed after weaning. Op is a recessive gene, toothless type occurs in obese parents, and toothless type occurs in the second generation. In normal cases, the maxilla and mandible have the main hair points of teeth. In osteosclerosis, there are hair points of growing teeth, but they cannot grow out because of hard bone. The pathological changes of this kind of mice are similar to those of human marbles.

Treatment methods include:

1. Parbiosis: It can cure some animals within one month.

2. Bone Marrow Grafts: This method is used for humans. The etiology of this disease is unknown, and the patient's hematopoietic function changes. This animal It can be used as a model to study human myelosclerosis.

Dominant mottle mutation anemia mice

(Dominant Spotting) Because the spot size refers to the size of white spotting, it is abbreviated as W. When it is normal, it is small w w, and when it is W w, it shows anemia. There are five alleles at W, and any two alleles can cause serious red blood cell Sexual and hypoplastic anemia can cause hepatic hematopoietic dysfunction at the same time. The severity of anemia is affected by environment and genotype. Different types of mutants can appear, such as W/+, W v/+, Wv/W v, W/W v, W/WJ, WJ, WN, etc. These mutants have no fertility and are black eyed and white.

W W can only survive for a few days after birth Modifier gene When it exists, Ww is partially dominant, with 90~98% white spots; If there is no modification gene, it will appear pure white, but it is black eye; If there are some modifying genes, different degrees of flecks may appear. W is a group of multiple alleles, for example, V of Wv means Viable. Wv Wv can live to adulthood, with white and black eyes, usually sterile, occasionally showing limited fertility, and its red blood cells are only half of normal, similar to human anemia.

For hematopoiesis system The mutant mice studied were:

1. Dominant Spotting mice

⑴ W/W v: hereditary anemia mice, which can live to adulthood, such as WBB6F1/J - W/W v mice.

⑵+/+: non anemia mice with normal red blood cell count, such as WBB 6F 1/J -+/+mice.

⑶ W/W: hereditary anemia mice with normal hematopoietic stem cells are mice with hematopoietic microenvironment defects.

⑷ W/W a: hereditary anemia mice with normal hematopoietic microenvironment are mice with hematopoietic stem cell defects.

2. an mutant (Hentwig's Anemia) mice.

Large red cell anemia. The severity of anemia varies with different genetic backgrounds. Hematopoietic function defect occurs on the 12th day of pregnancy, accompanied by leukopenia.

3. f mutant (Flexible Tail) mice:

High iron erythrocyte anemia, anemia changes due to the fetus liver Hematopoietic disorders cause anemia at birth. This kind of mice shows tail bending.

4. JO (Joundice) mutant mice, gestation Anemia occurs on the 14th day, jaundice occurs a few hours after birth, and death is often caused by brain damage, jaundice, bilirubinemia or hypoxia in the newborn period, Blood circulation Small reticular red blood cells and nucleated red blood cells were found in.

5. ha (HemolyticAnemia) mutant mice: hemolytic anemia in neonatal period, more than that in the 14th day of pregnancy. Neonatal jaundice, most of them die within a week after birth.

6. Sph (Spherocytosis) mutant mice: hemolytic anemia, spherocytosis bilirubin Hyperemia, death in a short time after birth.

7. Sla (Sex LinkedAnemia) mutant mice: mild anemia, reticulocytosis, and bone marrow deficiency. Growth of both sexes is blocked, but they can survive.

8. dm (Diminutive) mutant mice: dm/dm mice have small bodies, short and twisted tails, increased ribs and sacral anterior vertebrae, spinal deformity, costal segment fusion, and large cell anemia.

9. Ts (Tail Short) mutant mice: Ts/Ts mice often die in the uterus. By the eighth day of pregnancy, they show short and twisted tails and other bone abnormalities, prenatal anemia and yolk sac hemopoiesis disorders. Anemia can also lead to other abnormalities.

Cataract mutant mice

(Cat for short)

This is dominant inheritance, from 10 months to 14 weeks Lens opacification , will happen again Lens Liquefaction and super occlusion of the core. Cataracts occur when Cat is heterozygous. This character is easily observed in general, and can be used as an animal model of ophthalmology.

Spleen free mutant mice

(Asplenia, as for short)

The spleen of this mouse is completely absent, and it appears as a genetic disease in heterozygous mice with dominant hemilimb deformity. These mice have been widely used in spleen The study of function is also an important animal model for studying traditional Chinese medicine, and also a good experimental material for studying schistosomiasis.

Grey lethal mutant mice

(Grey Lethal, referred to as Gl)

The gl/gl mutant mice, in addition to being pure gray, are mainly characterized by growth disorders, which are smaller than normal 14 days after appearance. The teeth do not sprout, the shape is abnormal, and the teeth are not calcified. The long bones of limbs are abnormal. They usually die at the age of 22 to 30 days. Some people use their skeletal system disorders to study strontium metabolism.

Retinal degeneration mutant mice

(ReinalDegeneration, referred to as rd)

The relationship between the diseased mice and human Retinitis pigmentosa Similarly, human retinitis pigmentosa is also a genetic disease, presenting progressive retinal sclerosis, pigmentation and retinal vascular atresia and atrophy. It is difficult to obtain specimens for pathological research of human cases, while animal models provide extremely ideal experimental materials and can also be used for a series of other studies.

Desalination of lethal gene mutant mice

(Dilte Lethal, referred to as aL)

In addition to desalinization, dLdL also has convulsions and paralysis, and died about three weeks after birth. Later, it was found that the activity of phenylalanine hydrooxygenase in this disease was greatly reduced, so Model Humanoid Phenylketonuria 。

Needle tail mutant mice

(Pintail, Pt for short)

When Pt+is heterozygous, it shows dominant mutation characteristics, such as shortened and curled tails, and intervertebral discs degenerate rapidly from age to age, which is similar to that of human beings Prolapse of intervertebral disc , which can be used for this research.

Oligodactyly mutant mice

(Hypodactyly, Hd for short)

The rat has 2 toes on both forelimbs and 4 toes on both hind limbs. Oligodactyly occurs simultaneously in male rats sperm Lack (OIigospermia). Female rats have normal fertility. The coexistence of oligodactyly and sperm deficiency is an interesting phenomenon. Nude mice also have a similar situation. When hairless, fertility is low, and the relationship between them has not been clarified. Possibly associated with polymorphic genes or sexlinkage gene relevant. It is only found in mice and rats for unknown reasons.

Hypertensive mutant rats

（HypertensionRat）

Hypertensive rats are highly fertile and have a significant impact on life expectancy. They can be kept for 13-14 months. Okamoto has cultivated many sublines, of which SHR is the most valuable.

SHR (Spontaneously Hypertensive Rats), namely spontaneously hypertensive rats, was successfully bred by Okamoto and Aoki. Systolic blood pressure of normal rats 110-120mmHg, start to use ♂ 145-175mmHg× ♀ 130-140mmHg is used as the parent, and siblings will mate later. The blood pressure of the bred hypertensive rats can reach 150 mmHg 5 weeks after birth, while that of the adult male rats can reach 200 mmHg. Its characteristics are that the spontaneous rate of hypertension is 100%, the average blood pressure is 170-180 mmHg, and the maximum is 200 mmHg, and there are hypertensive cardiovascular diseases. Animals with high blood pressure (higher than 180mmHg) should be selected as breeding mice for each generation during reproduction.

Epileptic mutant rats

(AudiogeneicSeizures sound source seizure)

When the bell rings, it will rotate and dance for a few seconds, and then fall to the ground on one side epilepsy , similar to human epilepsy. This mouse can be used as an animal model to study human epilepsy. Physiology, Chinese Academy of Medical Sciences graduate school Both Beijing Medical University and Beijing Medical University feed 。

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