A transformant comprising MYH3 gene and use thereof

In the embodiment hereinafter the present invention through a corresponding business are provided as follows. In the embodiment of the present invention is generally described the present invention is to exemplify these for range and not the limited to those in the embodiment.

In the embodiment 1. Visual comparison of Pig meat transformation

Number of Pig meat ( and red meat) in order to compare a transformed making land race, from a free-form of his lampwick.

As a result, in Figure 1 the studied, but while introducing various sorts land race of mutton pale white, red and black evening twilight number making the mutton which has six, in particular excellent e [pul ring deposits has been confirmed.

The result of said, Pig compared to know capable of excellent quality for keeping things slicked land race.

In the embodiment 2. Pig meat transformation Quantitative characteristics sites(Quantitative trait locus; QTL) Analysis

In the embodiment 2 - 1. Identifying QTL gene region of meat

Pig meat for identifying modulators for determining gene traits, obtained as the number [lay pig and making a vaccine against porcine race land progeny (LK axis group), [lay pig and making a vaccine against porcine [tyu rock and number obtained as the transgenic progeny (DK axis group) where the meat for arthritis (red (a *) also, (IMF)) conducting quantitative characteristics sites (quantitative trait locus; QTL) analysis.

As a result, in the studied of Figure 2 a, vertical dashed lines is located on chromosome 12 times of LK axial army 661 kb formate, in addition, in a of Figure 2 b studied, even axial army vertical dashed lines is located on chromosome 12 times of 661 kb KD has been confirmed.

The result of said, red meat (red also) and determining the varietal Pig gene is damaged regions are positions have predetermined position, this chromosome 12 times of 661 kb know capable of existing in the data area.

In the embodiment 2 - 2. Pig meat identifying transformed gene

Pig meat once 661 kb of chromosome gene traits is determining said in the embodiment 2 - 1 through 12 is also used for by existing in the data area, said gene has been confirmed through a LALD mapping the existing in the data area.

As a result, in Figure 3 the studied, MYH3, MYH1, MYH2, MYH13, ADPRM, SCO1, TMEM220, ENSSSCG 00000029441, ENSSSCG 00000018006 9 gene is transformed by said gene has been confirmed that the meat of two existing in the data area.

In the embodiment 2 - 3. Pig meat and selecting transformed related gene

Periodically determining said in the embodiment 2 - 2 through 9 according to two meat traits gene, said gene to be among the most relevant gene was transformed aujesky determination of high quality.

(KNP) on land race (LANDRACE) (longest near, longissimus) and insides of the lampwick of Pig in (thigh company two muscle, quadriceps), two relative mRNA expression analyses the amount said 9 gene was.

Specifically, one race to the lampwick and insides of the Pig (LANDRACE) have a muscular tissues harvested on land, RNA (Ambion) was separating using Trizol reagent. Each organization TOPscript cDNA synthesis kit (Enzynomics) after 5 micro g such that the same RNA concentration by using cDNA (complement DNA) and copiers. After each organization qRT-a PCR using cDNA by conducting experiments. 95 °C qRT-a PCR experiments in 20 seconds, 20 seconds in 60 °C, in processing a 40 20 72 °C QuatiTect SYBR Green PCR kit (Qiagen) qRT-a PCR experiment for the implementation of a periodical repetition when the deflection, (Qiagen) equipment between embodiment was analyzed Roter provided Gene Q thermal cycler. Gene analysis of total 9 listed in table 1 was used for primer represented.

As a result, in a and b of Figure 4 a studied, compared to land race, and insides of the lampwick of MYH3 gene mRNA expression has been confirmed in Pig of highly crystalline.

The result of said, in particular transformed MYH3 gene among quality of swine, major gene determining red degree about his car.

In the embodiment 3. Pig MYH3 gene inserted with small number of transgenic mouse

In particular transformed through quality of swine MYH3 gene among said in the embodiment 2, by actually determining character that would yield a red gene, said gene activity in vivo (in vivo) in order to identify, a small 3 - 1 to 3 - 2 according to method for consecutively inserted with same number MYH3 gene transgenic mouse by using the air.

In the embodiment 3 - 1. Small number of vector

First, porcine MYH3 gene is inserted transgenic mouse number boil last summer, MYH3 gene expression vector inserted with CAGGS provided MYH3 provided Flag number was high pressure liquid coolant.

Specifically, overexpression of porcine MYH3 number for small, after confirming the entire bio-sequence listing of porcine MYH3 gene mRNA, DNA fragments is divided into a total 4 each artificially copiers. 1, 417Bp BglII enzyme XbaI site to both sites in cross-section and a first fragment thereof is in addition to the artificial copiers. A transparent conductive layer on the second fragment thereof is to synthesize 1, 745bp BglII SalI, SacII SalI synthesizing 1, 777bp on third fragment thereof is to have, on the second to last you 944bp SacII EcoRI-configurated. 4 DNA fragments bound to the enzyme site of finished end artificially using a porcine MYH3 gene mRNA including gene fragments (Ligation) coupled to finally complete the entire queue.

Also shown after overexpression vector for introducing 5 such as EcoRI XbaI cut CAGGS-a EGFP provided Puro vector and after, the number of the fragments prior work grudge porcine MYH3 gene mRNA transformed vector insert number was finally Pig MYH3 less complete (6 also).

The structure of the precursor to 6 said vector, vector nucleotide sequence sequence number 2 which have been designated.

In the embodiment 3 - 2. Small number of transgenic mouse

In the embodiment 3 - 2 - 1. Small number of transgenic mouse method

Said in the embodiment 3 - 1 transgenic mouse work grudge vector number through a small number-gate.

Specifically, C57BL/6n mouse fertilized egg (microinjection) after obtaining the microinjection technique using said transformed vector was introduced into it became work number to the nuclear embryo.

As a result, total 47 of F0 founder embryo therefrom.

In the embodiment 3 - 2 - 2. Through introducing foreign gene mRNA expression analysis for checking

Number through work grudge MYH3 gene have been introduced for whether said in the embodiment 3 - 2 - 1 transgenic mouse, mRNA expression of said gene was minisequencing.

Specifically, PCR experiment conditions in 30 seconds 95 °C, 60 °C in 30 seconds, when the periodical repetition in processing a 40 30 72 °C, represented using primer was listed in table 2.

As a result, studied in of Figure 7 a, is inserted in a transgenic mouse expressing MYH3 gene mRNA (21, 24, 26, 27, 28 times) were confirmed.

In the embodiment 3 - 2 - 3. Protein expression analysis for checking introduced foreign gene

Number through work grudge MYH3 gene have been introduced for whether said in the embodiment 3 - 2 - 1 transgenic mouse, his minisequencing protein expression of said gene.

Specifically, a transgenic mouse (TG) of insides of the wild-type mouse (WT) on muscle tissue after taking, RIPA buffer was added by spray-decomposing protein degradation billion number number ultrasound tissue. Then, through low temperature and protein in supernatant was harvesting the tissue are separated from each other. Protein assay reagent (bio-a rad) quantifying protein is BSA recovery after using, 4x protein loading buffer (1x) was about 10 minutes heating to 100 °C. In order to perform the [wey the [su it shook off using prepared protein, said protein SDS-a PAGE gel electrophoretic-gate about 2 hours. Then, moved from PVDF membrane after electrophoretic-protein, using 5% skim milk 1 have time blocking, stores an experiment first antibody (Anti-a Flag M2 (F1804, Sigma provided Aldrich) and b a-actin (#4970, Cell signaling)) has been added in reacting stored at 4 °C. Next TBST solution after 2 hours after washing the secondary antibodies reacting, ECL reagents have processing, (Fujifilm) LAS-a 300 luminescent image analyzer system using proteins of an antibody signal therefrom. Experiments used secondary antibodies as well as pace to the first antibody.

As a result, studied in of Figure 7 b, 24 times among the highest amount of protein expression in a transgenic mouse MYH3 gene were confirm it. The, highest degree protein expression after gene transformed mouse once it has been fruit 24 utilizing wave or vibrations, its appearance is also shown to 8.

In the embodiment 3 - 3. Identifying a transgenic mouse in the form of muscle

In order to identify the function of a crystal MYH3 gene transformed meat, meat of said in the embodiment 3 - 2 transgenic mouse number through work grudge traits visit from the police.

First, in a of Figure 9 a studied, porcine MYH3 gene is transformed in a wild type mouse (left; WT) mouse (shadow; TG) of muscle to the armrests of confirming capable of more than artificial pearl with red.

Then, in order to identify more particularly an muscle form, ATPase conducting dyeing. The wild type and a transgenic mouse (sucrose) 30% sucrose solution after processing to the armrests of muscles by recovering all night, OCT compound 10 using micro m in size then -25 °C tissue (tissue-a section), 4% PEA 1 was fixed during the time. After 10 minutes have flowing water cleaning, using 60% isopropanol (isopropanol) after washing, dyeing kit (ATPase stain lyopilized powder for histoenzymatic reaction kit, Bio optica yarn) ATPase in conducting method using irradiation number according to timing number database.

As a result, in a of Figure 9 b studied, as compared to wild-type mouse (left; WT), said gene transgenic mouse (shadow; TG) is inserted with the armrests muscles more red muscle relaxant is also used for distributed queue.

Additionally, in order to identify Oil Red O conducting dyeing of fat distribution in muscle tissue. Specifically, 0 said tissue fragments. 3% Oil Red O solution 1 with respect to the reaction time.

As a result, studied in of Figure 9 c, as compared to wild-type mouse (left; WT), is also used for dyeing a transgenic mouse (shadow; TG) degree by stronger, more muscle to the armrests of the fat content of a transgenic mouse were confirm it.

The same classifies, MYH3 gene is red and red muscle improve accumulate, gene cause that accumulates about his car.

In the embodiment 3 - 4. Of transgenic mouse intramuscular gene expression pattern checking

In the embodiment 3 - 4 - 1. Expression of gene formed near red/white muscle pattern checking

In order to identify the function of crystal quality MYH3 gene expressed in a transgenic plant, a transgenic mouse work grudge number through said in the embodiment 3 - 2 gene patterns of expression of red/white muscle of formed near visit from the police.

Specifically, a transgenic mouse (TG) on wild-type mouse (WT) collection of insides of the muscular tissues have, in conducting said in the embodiment 2 - 3 according to method qRT-a PCR. Selecting inner primer represented was listed in table 3.

In addition, the [wey the [su it shook off when the to perform said in the embodiment 3 - 2 - 3 according to method, selecting inner primary antibodies as follows: Anti-a Flag M2 (F1804, Sigma non-Aldrich), MYH7 (SC-a 53089, Santa cruz biotechnology), MYH4 (H00004622 - B01P, Abnova) and b a-actin (#4970, Cell signaling).

As a result, in a of Figure 10 a studied, as compared to wild-type mouse, a transgenic mouse of the same is slow type gene and protein expression of mRNA in red muscle in MYH7 gene has been confirmed by rapidly increased.

In addition, in a of Figure 10 b studied, as compared to wild-type mouse, electromagnetic wave is white muscle fast type gene in a transgenic mouse (Aldoa, Pvalb, Tnnf3, Tnnl2, Tnnc2) is a significant amount difference cannot be expression, red muscle slow type electromagnetic wave is affecting gene expression amount increases (Myoglobin, Tnnt1, Tnnl1, Tnnc1) are both mRNA has been confirmed.

In the embodiment 3 - 4 - 2. Fat accumulation pattern and related gene expression pattern checking

In addition, in order to identify the function of crystal quality MYH3 gene expressed in a transgenic plant, a transgenic mouse work grudge number through said in the embodiment 3 - 2 of fat accumulation pattern and related gene patterns of expression of visit from the police.

First, fat accumulation pattern form on tissue in order to identify whether numbers have, to this end H&E (Hematoxylin and eosin) conducting dyeing. Wild-type mouse (WT) and transgenic mouse (TG) using paraffin (Paraffin embedding) of muscular tissues after m hemp cloth [ting, a thickness of tissue fragment 4 micro m-gate. (Xylene) number using a stand-alone after xylene and paraffin, 100%, 95%, 75%, 50% alcohol in this order after the dehydration, washing water was flowing 5 minutes. Then, the [hey e [thok it was recorded Myyer's 1 minutes 20 minutes after processing (hematoxylin) solution flowing water washed and, it came to 1 minutes (esoin) after processing to carry out dehydration and clearing (clearing) have, on microscope after tissue dyeing state therefrom.

In addition, in order to identify patterns of expression of muscle tissue fat accumulation gene transgenic mouse, said in the embodiment 2 - 3 according to method qRT-a PCR is conducting. Selecting inner primer represented was listed in table 4.

As a result, studied in of Figure 10 c and d, as compared to wild-type mouse, intervals between which results in its purification of stem cell in a transgenic mouse, which deposited fat therebetween, in addition has been confirmed that the expression of fat differentiation gene significantly increases.

The result of said, red near related gene MYH3 gene is expressed in the hepatitis c viral replicon which improve gene as well as intramuscular fat formation causes about his car.

In the embodiment 4. MYH3 gene genotype analysis

MYH3 gene for analyzing the genotype, as starting point (transcription start site; TSS) from porcine MYH3 gene transcription of 5 '- UTR 3 kb, and from a stop codon (stop codon) 3' - UTR 1 kb bio-sequence listing was to decrypt.

As a result, in Figure 11 the studied, in meat QTN MYH3 gene affecting (i. E. , MYH3 provided _ 1805 provided 1810delGGACTG) has been confirmed (red point display). In addition, race number making [lay pig (KNP) on land (LANDLACE) between base mutation being regulated, this exon (exon) 3 (ATG) codon disclosure from the 5' - UTR located has been confirmed. When the number of the regions to identify defective becoming, - 1810 bp - 1805 bp making when, this has been confirmed by muscle style-modulators (myogenesis regulatory factor; MRF) binding sites.

In the embodiment 5. MYH3 gene of porcine password through the varietal determine genotype

Base mutation is present between said in the embodiment 3 MYH3 gene number making through land race and the identifying, using said base mutation was determines quality of Pig.

Specifically, primer (forward: 5 '- TGG TCT TTC CTA ATT GGT GAC AT-a 3' (seq ID no 65), reverse: 5 '- AGT TTT GAG CAA GGC TTT TGT T-a 3' (seq ID no 66)) was amplified using a base mutation of said portion. Pig DNA isolated from blood of 100 ng/micro l IIS facia, 10 × reaction buffer, 20 mm dNTP, 200 mm of each forward and reverse primer, 1. 5 Units Taq DNA polymerase (TaKaRa, Japan) 25 micro l capacity by adding sterile deionized water to conducting PCR reaction. PCR amplification is performed using PTC-a 200 (MJ Research, USA) have total 30 cycle, 60 °C the annealing temperature of the primer in the matter. Amplification product is 2% agarose gel containing EtBr (ethidium bromide) after electrophoresis on bitter cold, UV therefrom whether in gene amplification.

Then, cause bio-sequence listing of the variation in said base using HpyCH4IV number hanhyo small 'A▼CGT' his cutting portion. Said PCR amplification product (HpyCH4IV) have a number hanhyo small cutting using a, number hanhyo small reaction conditions according to the instruction of a provider of PCR product of 3 micro l 10X buffer 1 PCR on micro l, hanhyo small number 0. 3 Micro l, DW 5. 7 Micro l stored at 37 °C by mixing in his response. On 2% agarose gel containing EtBr (ethidium bromide) by electrophoretic, number by number making hanhyo small land race and cutting aspect [lay pig MYH3 gene has been confirmed.

As a result, in Figure 12 the studied, poor quality land race cut swine (LANDLACE) gene have not received in 250 bp band is also used for presented (1/1; non-truncated), [lay pig number making chopped meat is excellent (KNP) gene has been confirmed that the two band as indicated by 77 bp and 167 (2/2; truncated). In addition, the land race number has been confirmed that the two band as indicated by 250 and 167 bp making fruit varieties (1/2; truncated).

The result of said, MYH3 gene traits of Pig meat in determining the possibility of foreign of Pigs and health by porcine it may be as well as, through Pig has been confirmed that the quality can be determined.

Description of or more from, the present invention of the present invention is provided to the one skilled technical idea or essential characteristics thereof without changing other specific embodiment can form can be understand are disclosed. In this regard, in the embodiment described above are exemplary in all of which must understand not definitive provided therein. The meaning of the description of the present invention carry patent said range rather than if all of the form of the present invention derived from equivalent general outline and range and method for changing or modified range should interpreted.