CN104342485A - Application of Isl1 gene or protein in sinus node abnormality related diseases - Google Patents

Abstract

The invention provides application of Isl1 gene or protein in sinus node abnormality related diseases. Specifically, the invention provides an application of LIM homeodomain transcription factor gene or protein thereof in preparing a reagent or a kit for detecting or predicting a sinus node (SAN) abnormality related disease. The invention also provides a method for detecting or predicting sinus node abnormality related diseases. The present invention can be used for prenatal screening and diagnosis of various congenital heart diseases with arrhythmia as a main symptom, and is favorable for prenatal and postnatal care.

Description

Isl1 gene or the application of albumen in the abnormal relative disease of sinus node

Technical field

The present invention relates to congenital heart disease diagnoses and treatment field, particularly, the present invention relates to Isl1 gene, the application of albumen in the abnormal relative disease of diagnosis sinus node.

Background technology

Cardiac pacing conducting system is a kind of special cardiac muscular tissue, and it produces and propagates electricimpulse, plays an important role in the Rythmic contractions characteristic of regulation and control heart.Pace-making conducting system is grown and the exception of function can cause irregular pulse and sudden cardiac death.

In China, have every year, more than 500,000 examples, sudden cardiac death occurs.And irregular pulse is the first cause of sudden cardiac death.

Cardiac pacing conducting system saves (AVN) by pacemaker (sinus node/SAN), chamber and Purkinje fiber forms.Mouse, flow into road at embryo's the 8th day (E8.0) heart and start to be recorded to cardiac pacing signal.Embryo the 9.5th day (E9.5), the venous sinus (sinus venosus) that heart flows into road is original pacemaker areas.And morphologic pacemaker is formed at E11.5, ripe further thereafter, be formed as a merit can completely pacemaker.The formation of pacemaker is the process of complicated, a meticulous adjustment, relates to the cardiac linage of multiple different sources.

In recent years, the research of the molecular regulation mechanism that conducting system of heart is formed is made remarkable progress.Research shows, derive from ventricular endocardium and Endothelin-1 coronarius Purkinje fiber differentiation-inducing in play critical effect.Notch and Neuregulin signal path plays a significant role in the formation of AV (room-room) conducting system of zebra fish.In heart and cardiomyocytes cultured, Neuregulin-1 and Endothelin-1 can inducing mouse Cardiomyocyte Differentiation be pace-making conducting system cell.Transcription factor Nkx2.5, Tbx5 and GATA4 interact, and form an important cardiac transcription regulated and control network, in the growth of heart, comprise in the formation of AV conducting system and function and playing an important role.The variation of these factors result in human congenital's heart trouble, abnormal with AV conducting system.

But comparatively speaking, the a large amount of Deaths of embryo can be caused with the disappearance of pace-making genes involved due to known, pacemaker cell is also difficult to differentiate in weave construction, and pacemaker cell limited amount, be difficult to separation and purification, cause to cardiac pacing conducting system in prior art, particularly very limited to the understanding of Cardiac pacing cell formation and function point analysis mechanism.

Therefore, in order to understand mankind's irregular pulse genesis mechanism in depth, contributing to prenatal and postnatal care and providing treatment means, this area grows the gene relevant with function and regulatory pathway thereof in the urgent need to exploitation to cardiac system.

Summary of the invention

Object of the present invention is just to provide a kind ofly grows the gene relevant with function and regulatory pathway thereof and application to cardiac system.

The invention discloses a kind of LIM homologous structure domain transcription factor (LIM homeodomain transcription factor, Islet1, Isl1) purposes of gene or its albumen, for the preparation of the reagent or the test kit that detect or predict sinus node (SAN) abnormal relative disease.

In another preference, described Isl1 gene comprises Isl1 genome sequence, cDNA sequence or mRNA sequence.

In another preference, described Isl1 gene or albumen come from Mammals, more preferably derive from rodent (as mouse, rat) or primate (as people).

In another preference, described Isl1 gene or dietary protein origin are in people.

In another preference, the nucleotide sequence of described Isl1 gene is as shown in SEQ ID NO.:1, and the protein sequence of its coding is as shown in SEQ ID NO.:2:

In another preference, the abnormal relative disease of described sinus node comprises: sick sinus syndrome or conducting system of heart are correlated with irregular pulse.

In another preference, described conducting system of heart is correlated with that irregular pulse comprises atrial fibrillation, room is flutterred, Fang Zao, room morning, preexcitation syndrome, sinus arrhythmia.

In another preference, described reagent comprises Isl1 Auele Specific Primer, specific antibody, probe and/or chip.

In another preference, described detection comprises enzyme linked immunoassay method (ELISA method) and detects or Time-resolved Fluoimmunoassay (TRFIA method) detection.

In another preference, described Isl1 albumen or its specific antibody coupling has or with detectable label.

In another preference, described detectable label is selected from lower group: chromophore, chemiluminescent groups, fluorophore, isotropic substance or enzyme.

In another preference, the specific antibody of described Isl1 is monoclonal antibody or polyclonal antibody.

In another preference, described detection measures amniotic fluid samples, tissue sample, blood sample, serum sample or humoral sample.

In another preference, described detection measures amniotic fluid samples.

In another preference, described amniotic fluid samples is from mouse E8-E14 or the mankind embryo of 12 weeks 20 weeks.

In another preference, described amniotic fluid samples is from mouse E9.5-E11.5 or the mankind embryo of 4 weeks 12 weeks.

In another preference, described reagent comprises:

A () detects the nucleic acid chip of Isl1;

The specific antibody of (b) anti-Isl1 albumen; And/or

The genomic dna of (c) specific amplification Isl1, the Auele Specific Primer of mRNA or cDNA.

Second aspect present invention, provides a kind of test kit for detecting or predict the abnormal relative disease of sinus node, and described test kit comprises and detects the nucleic acid chip of Isl1 or the Auele Specific Primer of specific amplification Isl1 nucleotide sequence and specification sheets; Wherein said specification sheets comprises following content: detected by the expression and/or activity that measure Isl1 gene or albumen or predicted sinus node (SAN) abnormal relative disease.

In another preference, Isl1 nucleotide sequence comprises genome sequence, mRNA sequence or cDNA sequence.

In another preference, described detection measures amniotic fluid samples, tissue sample, blood sample, serum sample or humoral sample.

In another preference, described detection measures amniotic fluid samples.

In another preference, following content also recorded by described specification sheets: amniotic fluid samples is from mouse E9.5-E10.5 or embryo's amniotic fluid in 4 weeks-12 weeks of the mankind.

In another preference, following content also recorded by described specification sheets: detect the required Isl1 gene of object or the expression of albumen and/or activity, if significantly lower than normal population, then illustrate, this detected object suffers from the probability of the abnormal relative disease of sinus node higher than normal population.

In another preference, described test kit is also containing one or more reagent being selected from lower group:

(i) for extracting the reagent of amniotic fluid or cells in sample genomic dna or RNA;

(ii) for the primer of reverse transcription;

(iii) for the ThermoScript II of reverse transcription reaction;

(iv) for the polysaccharase of PCR reaction.

In another preference, described detection is that real-time fluorescence quantitative PCR detects.

In another preference, described detection measures amniotic fluid samples.

Third aspect present invention, provides a kind of method detecting or predict the abnormal relative disease of sinus node, comprises step:

A () measures the required Isl1 gene of object or the expression of albumen and/or activity;

(b) by the measurement result that obtains in (a) compared with the Isl1 gene of normal population or the expression of albumen and/or activity;

Wherein, if the expression of the Isl1 gene of required object or albumen and/or activity are significantly lower than normal population, then illustrate, this detected object suffers from the probability of the abnormal relative disease of sinus node higher than normal population.

Fourth aspect present invention, provides a kind of method preparing animal model, comprises step:

Genotypic for Isl1hypo/+ rodent and the genotypic rodent of Isl1+/nLacZ are carried out mating or genotypic for Isl1hypo/+ rodent and the genotypic rodent of Isl1hypo/+ are carried out mating, the rodent filial generation thus the acquisition low expression of Isl1 compound (Isl1hypo/nLacZ) makes a variation.

In another preference, described rodent comprises Mouse and rat.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

Fig. 1 shows Mouse Embryo Development and the rear expression of Isl1 in sinus node (SAN) pacemaker cell of birth

E9.5 days, Isl1 and HCN4 are expressed in venous sinus (SV) myocardium (Figure 1A) simultaneously, are then mainly expressed in (Figure 1B-E, Fig. 1 I) in SAN cell in E10.5 to P7 sky.At E10.5 days, Tbx3 and Isl1 coexpression in SV, comprise region end to end and the venous valve (vv) (Fig. 1 F, G) of SAN.Cx40 is expressed in atrial myocardium, with Isl1 coexpression (Fig. 1 H, J).K value in the drawings in boxed area is as shown in Fig. 1 I and Fig. 1 J.The expression of Isl1 is (Fig. 1 L) that reduce gradually in Mouse Embryo Development and postnatal process.

The expression that Fig. 2 shows Isl1 reduces the minimizing causing embryo's irregular pulse and SAN cell

Isl1-nLacZ is expressed in SV myocardium, comprises SAN region (red arrow), E9.5(Fig. 2 A, A') and E11.5(Fig. 2 D, D') dorsal mesoderm and mesocardium.The expression that Isl1 and HCN4 suddenlys change in mouse SV at ISL1 significantly reduces (Fig. 2 C, C').The expression of Isl1 and Isl1 positive cell, at ISL1 sudden change mouse E9.5 (Fig. 2 B, B') and E11.5 (Fig. 2 E, E') embryo SV myocardium, comprise SAN (red arrow), significantly reduce in atrial myocardium and mesocardium.The cell proliferation of BrdU dyeing display ISL1 sudden change mouse E9.5 embryo SV myocardium significantly reduces (Fig. 2 F, F').TUNEL dyeing display ISL1 sudden change mouse E10.5 embryo SV apoptosis significantly increases (Fig. 2 G, G').Ultrasonic cardiogram result shows that ISL1 sudden change mouse E9.5 and E11.5 embryo heart rate significantly reduce (Fig. 2 H).

Fig. 3 shows the HCN4 of ISL1 sudden change mouse embryo SAN, Tbx3 and Shox2 expresses reduction

HCN4 and Shox2 is expressed in mice embryonic E9.5 days SV, comprises SAN(Fig. 3 A, A', B, B', red arrow).Tbx3 is expressed in the mesenchymal cell (Fig. 3 C, C', arrow) around SV.Isl1 sudden change mouse embryo, in SV and SAN, the expression of HCN4, Shox2 and Tbx3 all significantly reduces (Fig. 3 F, F', G, G', H, H', red arrow).Cx40 and Nkx2.5 expresses at myocardium, is not expressed in (Fig. 3 D, D', E, E', red arrow) in SAN.Isl1 sudden change mouse embryo, Cx40 and Nkx2.5 significantly reduces in atrial myocardium, but Cx40 and Nkx2.5 there is no ectopic expression (Fig. 3 I, I', J, J', red arrow) in SAN.

Fig. 4 shows embryo's early application HCN4-CreERT2 and in SAN, knocks out Isl1 irregular pulse and SAN cell quantity can be caused to reduce

Isl1 sudden change mouse (HCN4-CreERT2; And control group (HCH4-CreERT2 Isl1f/f); Isl1f/+or+ /+) embryo gives tamoxifen in E9.5 gavage, within 32 hours and 48 hours, analyzes embryonic phenotypes upon administration.Open in-heart operation under pulsating display Isl1 suddenlys change mouse embryo at E11 days than E11.5 days hearts rate reduction more obvious (Fig. 4 A).The quantity (Fig. 4 C, c1, c2, red arrow and D) that overall and section X-gal dyeing is presented at X-gal positive cell in E11.5 days Isl1 sudden change mouse embryo SAN is less than control group (Fig. 4 B, b1, b2, red arrow).And the quantity of HCN4 positive cell in E11 days sudden change mouse embryo SAN regions is without obviously changing (Fig. 4 E, F, red arrow).Isl1 suddenlys change in mouse (E11), and Isl1, HCN4 and Tbx3, in the expression of SV, comprise SAN region (red arrow).Isl1 mouse (Fig. 4 H, J, L) relative comparison group (Fig. 4 G, I, K) of suddenling change has and significantly to weaken but Isl1 is normal at pharyngeal and expression amount that is dorsal mesocardium region.

Fig. 5 shows and knocks out Isl1 late period in SAN morphological development and can cause bradyrhythmia, Tbx3 and HCN4 expresses reduction in SAN

Knocking out Isl1 at E11.5 days can cause mutant mice to occur that heart rate reduces (Fig. 5 A) at E12.5 and E14.5.In fetal development, embryo heart rate increases (Fig. 5 B) gradually with fetal development.When Isl1 is after E13.5 embryo knocks out, use cardiac ultrasonic monitoring mutant mice heart rate, have no obvious change at initial 24 hours mutant mice hearts rate, but after 24h, Isl1 mutant mice occurs that heart rate reduces gradually.Entirety and X-gal positive cell (Fig. 5 D, D') in section X-gal dyeing display Isl1 mutated embryonic SAN comparatively control group embryo (Fig. 5 C, C') slightly reduce.Quantitative analysis shows, X-gal positive cell quantity in Isl1 mutated embryonic SAN region (175.5 ± 11.7/ sections, each SAN counts 10 sections) (D') comparatively control group (205.6 ± 17.6/ cut into slices) (Fig. 5 C') reduces 15%(Fig. 5 E-H).Isl1, HCN4 and the Tbx3 expression in Isl1 mutated embryonic reduction (Fig. 5 F, H) remarkable in control group embryo (Fig. 5 E-G).

Fig. 6 shows the reduction knocking out Isl1 and cause the expression of Cell cycle-related genes, transcription factor and ionic channel.

QPCR display (Fig. 6 A) knocks out Isl1 and causes the expression of Cell cycle-related genes and Bcl-2 to reduce; Shox2, Mef2C, Tbx3 and Nkx2.5 express and reduce (Fig. 6 B); A series of ionic channel, the expression of connexins and Myh6 changes (Fig. 6 C).Mouse and mankind Tbx3 and HCN4 genome area comparison diagram (E1-7: exon is blue; Uncoded district is yellow; Conservative region is red and pink) (Fig. 6 D, E).Nano-ChIP method detects 2 Isl1 respectively at Tbx3 (Tbx1-1 ,-2) and HCN4(HCN4-1 ,-2) possible binding site.QPCR analysis verification Nano-ChIP method detect 2 Isl1 respectively at Tbx3 (Tbx1-1 ,-2) and HCN4(HCN4-1 ,-2) binding site (Fig. 6 F).

Fig. 7 shows cTnT-Cre and knocks out Isl1 mouse (cTnT-Cre; Isl1 mouse is) and the phenotype of Isl1 compound sudden change mouse similar, this sudden change mouse is in the embryonic stage of i.e. E9.5-E11.5 death in early days, and mutated embryonic shows the slack-off and premature beat of obvious heart rate.

Fig. 8 A-Fig. 8 I shows in the SAN region of expressing at HCN4, and the cell quantity of TUNEL mark significantly increases, and the cell quantity of BrdU mark reduces (arrow place).

Embodiment

The present inventor is through extensive and deep research, overcome the technical barrier that the early stage Isl1 genetically deficient of fetal development causes animal model mortality first, establish the mouse model that the low expression of Islet1 and condition knock out, through experimental studies have found that this expression model, at fetal development middle and advanced stage, the low expression of Islet1 can result in serious decreased heart rate or arrhythmia, and this kind of embryo can be survived mostly to birth.

Experiment proves, even if Isl1 gene wide expression is during fetal development, but its early expression and middle and advanced stage express the regulating and controlling effect that rises different, the congenital cardiac arrhythmia that embryo's middle and advanced stage low expression Isl1 can cause birth filial generation serious, the embryonic death that the low expression of the more early stage Isl1 of this consequence or disappearance cause, can bring larger misery and burden for family and society.

In addition, the present invention have also demonstrated, and Isl1 gene, as the upstream gene of HCN4 path, in the middle and advanced stage of pacemaker cell is grown, serves important regulating and controlling effect.On this basis, the present invention is completed.

Term

As used herein, in the formation that term " the second cardiac field " refers to heart and growth course, there is part cardiac precursors to derive from front dorsal part mesoderm, and this region is defined as the second cardiac field.

Term " the second cardiac field precursor cell " refers to that deriving from the second cardiac field can become the cell mass of ripe heart cell by differentiation and development further.

Term " pacemaker precursor cell " refers to can become the cell mass of ripe pacemaker cell by differentiation and development further.

As used herein, term " sinus node abnormal " refers under pathology or physiologic factor, and the pace-making autonomy of sinus node cells and rhythmicity are affected the sinus node dysfunction caused, and cause the situation of related pathologies symptom.Sinus node is extremely usual to be caused by congenital factor and autonomic nerve influence factor etc.

As used herein, term " sinus node abnormal relative disease " refers to sinus node pace-making and conduct relevant irregular pulse.The abnormal relative disease of usual sinus node comprises sick sinus syndrome or conducting system of heart and to be correlated with irregular pulse.

As used herein; term " conducting system of heart be correlated with irregular pulse " refers to directly or indirectly to affect due to sinus node exception or functional defect cardiac rhythm and conduction and causes ARR pathologic conditions, comprise atrial fibrillation, room flutterred, Fang Zao, room morning, preexcitation syndrome, sinus arrhythmia.

As used herein, term " Cre ", " Cre enzyme " are used interchangeably, and all refer to Cre recombinase, are the I type topoisomerase of bacteriophage P1, carry out Site-specific recombinase for the DNA between catalysis loxP site.In the present invention, be mainly used in knocking out the specific gene between LoxP site.

As used herein, term " fetal development is early stage " refers to that early embryonic development comprises some stages such as fertilization, the spilting of an egg, blastaea and implantation, gastrula and germinal layer formation, differentiation of germinal layers and the formation of idiosome embryo.Corresponding rodent fetal development is E7.5-E11.5 in early days, and it is the 1-3 month that corresponding human embryos are grown in early days.

As used herein, term " fetal development middle and advanced stage " refers to that almost all histoorgan was formed with ripe until the period of birth.Corresponding rodent fetal development middle and advanced stage and E12.5-E21, it is the 4-9 month that corresponding human embryos grow middle and advanced stage.

Term " Tbx18 pedigree ": Tbx18 transcription factor is expressed in proepicardium cell and epicardial cell, important regulating effect has been formed to the growth of heart, therefore can specificity marker heart, and the proepicardium cell of heart and epicardial cell pedigree be defined as Tbx18 pedigree.

As used herein, term " the compound mutant mice of Isl1 " refers to the compound sudden change mouse of Isl1 (Isl1nLacZ/f ^neo), namely pass through Isl1f ^neothe mouse model that /+mouse system and the hybridization of Isl1nLacZ/+ mouse system obtain.

" tamoxifen " (tamoxifen) is a kind of exogenous estrogen analogue, the estrogen receptor in chimeric recombinase can combine and make Cre enter nucleus and LoxP site is combined to knock out specific gene.

Term " Tbx18 pedigree ": Tbx18 transcription factor is expressed in proepicardium cell and epicardial cell, important regulating effect has been formed to the growth of heart, therefore can specificity marker heart, and the proepicardium cell of heart and epicardial cell pedigree be defined as Tbx18 pedigree.

Islet1

Isl1 gene source is in No. 5 chromosome long arm, and the about 2.4kb of total length, 349 amino acid of encoding, derives from encoding sequence (Genbank accession number BC132609.1), Protein Information (Genbank accession number AAI32610.1)

Islet1 is a homologous structure domain transcription factor, whole body knocks out disappearance completely and the embryo's Deaths (E9.5-10.5) that Islet1 causes the cardiac structure deriving from Islet1 precursor cell, which prevent the further research acted in pacemaker Islet1.

Isl1 of the present invention has the homology of height in several species, and Isl1 used in the present invention can come from any Mammals, comprises (but being not limited to): people, rodent (as mouse, rat), chimpanzee, ox, dog, cat etc.In addition, Isl1 comprises wild-type Isl1 and saltant type Isl1 and active fragments thereof.

HCN4

HCN4(hyperpolarization activity cyclic nucleotide gate passage 4, hyperpolarization-activated cyclic nucleotide-gated channel4) gene source is in Chromosome 9, mRNA total length 3.7kb, to encode 1201 amino acid, its nucleotide sequence is (gene ID NM_001081192.) as shown in SEQ ID NO.:3, and the protein sequence of its coding is as shown in SEQ ID NO.:4 (NP_001074661.1).

Just express at heart crescent phase (Cardiac Crescent) HCN4.In growth period and one-tenth human heart, HCN4 indicates sinus node specifically.

The mankind, the variation of HCN4 causes bradycardia.In the animal patterns such as mouse, knock out HCN4 completely and cause serious frequent sinus heartbeat to stop fighting, and embryo's Deaths (E9.5-10.5).But knock out HCN4 at adult mouse heart and do not cause dead mouse, the basal heart rate/rule of mutation is normal, but has intermittent cardiac arrest.These researchs show that HCN4 is required to early stage heart pace-making function.HCN4 and calcium current clock (Calcium clock) determine the high autorhythm of pacemaker cell, in the adjustment of the autorhythmic generation of the sinus of heart and HR Heart Rate, play keying action.In the Adulthood, other mechanism comprises calcium current clock and relevant Function protein may play a role jointly.

Detection method and test kit

The invention still further relates to quantitative and qualitative detection people Isl1 protein level or mRNA level in-site diagnostic testing process.These tests are known in the art.The people Isl1 protein level detected in test, may be used for detecting or the abnormal relative disease of prediction sinus node.

A kind of method that whether there is Isl1 albumen in sample that detects utilizes the specific antibody of Isl1 albumen to detect, and it comprises: contacted with Isl1 protein specific antibody by sample; Observe and whether form antibody complex, define antibody complex and just represent in sample to there is Isl1 albumen.

Isl1 albumen or its polynucleotide can be used for the Diagnosis and Treat of Isl1 protein related diseases.Part or all of polynucleotide of the present invention can be used as probe and is fixed in microarray or DNA chip, for analyzing Differential expression analysis and the gene diagnosis of gene in tissue.The antibody of anti-Isl1 can be fixed on protein chip, for detecting the Isl1 albumen in sample.

Present invention also offers a kind of test kit detecting the abnormal relative disease of sinus node, it contains the primer pair of specific amplification Isl1 and/or Isl1 specific antibody and label or specification sheets.

Wherein, described label or specification sheets indicate following content: the expression of Isl1 gene or albumen and/or activity significantly lower than normal population, then illustrate, this detected object suffers from the probability of the abnormal relative disease of sinus node higher than normal population.

The inventive method and test kit can be used for detecting people's amniotic fluid samples, tissue sample, blood sample, serum sample or humoral sample, for the abnormal relevant congenital cardiac arrhythmia of early screening sinus node, are conducive to prenatal and postnatal care.

Promotor and pharmaceutical composition

Utilize albumen of the present invention, by various conventional screening assays, can filter out, with Isl1 albumen, interactional material occur, especially promotor etc.

The promotor of Isl1 albumen of the present invention, when carrying out using (administration) on treating, can promote expression and/or the activity of Isl1 albumen, and then promotes HCN4 gene or protein expression, or activates transcribing of HCN4.In another preference, the Isl1 gene expression product that described Isl1 promotor comprises, promoted type microRNA, promoted type transcriptional regulator or promoted type target micromolecular compound.

Usually, these promotor can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can with being formulated the character of material and illness to be treated and changing to some extent.The pharmaceutical composition prepared can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Present invention also offers a kind of pharmaceutical composition, it contains the Isl1 albumen of the present invention of safe and effective amount or its promotor and pharmaceutically acceptable carrier or vehicle.This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.The pharmaceutical composition of such as Tablet and Capsula and so on, is prepared by ordinary method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should aseptically manufacture.The dosage of activeconstituents is treatment significant quantity, such as every day about 1 microgram-10 mg/kg body weight.

Universal method

Transgenic mice

The structure of Isl1-nLacZ gene knock-in mouse (being designated as Isl1nLacZ/+) is by ordinary method, mouse 1 to chromosomal wherein one in Isl1 initiation site insert nLacZ, a kind of preferred visible Song of construction process, M.R., Sun, Y., Bryson, A., Gill, G.N., Evans, S.M., and Pfaff, S.L.2009.Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity.Development136:2923-2932.In this mouse, the Isl1 of item chromosome can not normal expression, and another item chromosome can normal expression Isl1.

TnT-Cre mouse system (being designated as cTnT-Cre), cTnT-Cre by fix a point the method knocked in by mouse 1 to chromosomal wherein one after the initiation site of cTnT, insert recombinase gene cre, Cre by the regulation and control of cTnT promotor only at the Expression in Myocardium of differentiation.In this mouse, item chromosome can express recombinase gene cre.This mouse is purchased from south, Shanghai model animals Science and Technology Ltd..

Jiao is shown in by the structure of Isl1floxed mouse system, K., Kulessa, H., Tompkins, K., Zhou, Y., Batts, L., Baldwin, H.S., this mouse of and Hogan, B.L.2003.An essential role of Bmp4in the atrioventricular septation of the mouse heart.Genes & development17:2362-2367. system replaces with two ends four exon with LoxP site mouse 1 to chromosomal Isl1 the 4th exon by the method for homologous recombination.

CTnT-Cre; Isl1 mouse is: be obtained by cTnT-Cre mouse system and the hybridization of Isl1floxed mouse system.Hybridize mice can be expressed recombinase gene Cre and can be identified the loxp site of inserting Isl1 the 4th exon two ends thus be deleted by Isl1 the 4th exon, makes Isl1 gene cannot normal expression.This mouse is that cTnT-Cre mouse system and the hybridization of Isl1floxed mouse system obtained.

Isl1 is low, and expression (is designated as Isl1f ^neoisl1 the 4th exon to be replaced with the 4th exon with loxp site and screening-gene neo by homologous recombination by/the+) structure of mouse system.A kind of preferred visible Sun of construction process, Y., Dykes, I.M., Liang, X., Eng, S.R., Evans, S.M., and Turner, E.E.2008.A central role for Islet1in sensory neuron development linking sensory and spinal gene regulatory programs.Nature neuroscience11:1283-1293. neo in this mouse can disturb the allelic normal expression of the Isl1 at neo place, because the compensation Isl1 of other item chromosome expresses normal in chimeric mice; But in pure and mild mouse, make the expression level of Isl1 significantly reduce, death in p1 days after birth.

Tamoxifen induction type HCN4-Cre ERT2(is designated as HCN4-CreERT2) mouse builds available homologous recombination technique and carries out.A kind of preferred method is that the cDNA segment one being comprised CreERT2 sequence is inserted at the position near HCN4 gene start codon (ATG), builds transgenic animal model.A kind of preferred visible Liang X of construction process, Wang G, Lin L, Lowe J, Zhang Q, Bu L, Chen YH, Chen J, Sun Y, this mouse of Evans SM.HCN4Dynamically Marks the First Heart Field and Conduction System Precursors.Circ Res.2013Jun6.Epublication wherein item chromosome can express CreERT2, and another expresses HCN4.

The compound sudden change mouse of Isl1 (is designated as Isl1nLacZ/f ^neo): by by Isl1f ^neo/+mouse system and the hybridization of Isl1nLacZ/+ mouse system obtain.Mouse 1 pair of karyomit(e) that hybridization obtains, wherein one is inserted nLacZ at Isl1 initiation site, makes Isl1 can not normal expression.In another item chromosome Isl1 gene intron in insert neo gene, interference Isl1 expression.

HCN4-CreERT2/Rosa-LacZ, YFP mouse: HCN4-CreERT2 mouse system is hybridize with the Isl1flox/flox mouse with Rosa-LacZ or YFP genetic background, to obtain HCN4-CreERT2/Rosa-YFP or LacZ mouse.This mouse can be expressed the loxP site in the middle of CreERT2 identifiable design Rosa-YFP or Rosa-LacZ and its middle gene order be deleted, and is the expression that Rosa can start YFP or LacZ.

The Cre enzymic activity be to induce HCN4-CreERT2 mouse, gives 150-300ul tamoxifen (10mg/ml) to the female mouse of different brephic pregnancies required time point is oral.Early stage to embryo, tamoxifen is in mice embryonic E9.5 administration, and embryo samples is 32-36 hour upon administration, about gathers in the crops E11 days embryonic stage.To late embryo stage, tamoxifen is in mice embryonic E11.5 administration, and embryo samples is about gathered in the crops upon administration E14.5 days embryonic stage.The enable expression observing HCN4-CreERT2 after tamoxifen induction Cre expresses of the Rosa26-YFP genetic background that transgenic mice has.

Immunofluorescence dyeing and RNA in situ hybridization

X-gal dyes, and the method using conventional procedures of immunofluorescence and RNA in situ hybridization carries out, and representational method is visible:

Liang,X.,Zhou,Q.,Li,X.,Sun,Y.,Lu,M.,Dalton,N.,Ross,J.,Jr.,and Chen,J.2005.PINCH1plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.Molecular and cellular biology25:3056-3062；

Liang,X.,Sun,Y.,Schneider,J.,Ding,J.H.,Cheng,H.,Ye,M.,Bhattacharya,S.,Rearden,A.,Evans,S.,and Chen,J.2007.Pinch1is required for normal development of cranial and cardiac neural crest-derived structures.Circ Res100:527-535。

Use primary antibodie is as follows: mouse anti-Islet1/2 monoclonal antibody (DSHB), rabbit anti-Islet1(abcam), rat anti-HCN4(abcam), rabbit anti-Cx40(Santa Cruz), sheep anti-Tbx3(Santa Cruz), mouse anti-Nkx2.5(Santa Cruz), rat anti-Brdu(abcam).Two anti-employ Alexa488or594labeled(Invitrogen).TUNEL staining employs kits manuals (purchased from Roche).

BrdU staining: pregnancy female mouse abdominal injection 500 μ l BrdU (premix liquid of particular point in time, Ambion) 3 times, 3 hours, interval, BrdU dyeing is undertaken by ordinary method, representational method is shown in Sun, Y., Dykes, I.M., Liang, X., Eng, S.R., Evans, S.M., and Turner, E.E.2008.A central role for Islet1in sensory neuron development linking sensory and spinal gene regulatory programs.Nature neuroscience11:1283-1293.

Cell counting: to specific budding heart SAN region samples section, slice thickness 10um, each sample is chosen 4 serial section and carried out dyeing and counting positive cell.In cell proliferation experiment and cell apoptosis assay, to the BrdU in venous sinus and SAN region on the right side of mutated embryonic and contrast embryo, positive and TUNEL positive cell counts.In order to study the expression of Isl1 in growth course, the Isl1 positive and HCN4 positive cell are counted, in order to calculate the ratio of Isl1 and HCN4 positive cell.To the different samples of each time point, the section that the minimum analysis of each sample 4 to 6 is corresponding, the minimum analysis of each experiment 3 pairs of samples.

Nano-ChIP and real-time quantitative PCR (qPCR):

Nano-ChIP of the present invention and real-time quantitative PCR (qPCR) are undertaken by ordinary method, and representational method is as follows:

Adopt FACS-sorted method, be separated HCN4-CreERT2; Isl1f/f sudden change mouse embryo and HCN4-CreERT2; Isl1 control group embryo (Rosa-YFP background) SAN pacemaker cell is used for qPCR and analyzes.Gather in the crops and perform the operation the Isl1 sudden change and control group embryo SANs that are separated E11 days YFP positives, use collagenase/dispase(1mg/ml, Roche)/trypsin(0.1%) mixture slaking liquid digests 10 minutes.Collect the pacemaker cell (YFP is positive) be suspended in pace-making Digestive system, FACS sorted(BD FACSAria) isolated cell be stored in-80C.RNA uses RNeasy Mini kit(Qiagen) preparation.QPCR uses SYBR green method.

Nano-ChIP experimental technique: HCN4-nEGFP embryo SAN cell collects (E13.5to E18.5) at late embryo stage, and cell harvesting and FACS-sorted method are as previously mentioned.About 40,000SAN cells use 1%formaldehyde at normal temperature crosslinked 10 minutes.Cell uses glycine cold quenching 5 minutes and rinses 2 times with frozen water precooling PBS.Chromatin uses ultrasonic wave to be fragmented into 200-800bp size segment, and uses 1 μ g Isl1 antibody (39.4D5, DSHB) co-precipitation.Oppositely be cross-linked subsequently and purifying, the DNA of ChIP DNA and input is amplified (60).After amplification, DNA uses qPCR qualification, and associated regulatory region is by confirming after analytical data.

Ultrasound cardiograph detects:

Pregnant mouse uses isoflurane anesthesia.Use ultrasound cardiograph model for Visualsonics Vevo770 high-res ultrasonic system, use RMV704 (40MHz) probe.Each embryo detects separately, and obtains independently image and data.Heart rate and every heart function parameter is obtained by acquisition B-mode and pulse Doppler image.After ultrasound cardiograph detects, solution cuts embryo, corresponding with ultrasound cardiograph detected result respectively, and carries out genotype and phenotype detection.

Statistical analysis method

All data use mean value (mean) ± SEMb to represent, t-test uses 2 groups of independent test results mutually to verify.Think that experimental result has significant difference as P<0.05.

Beneficial effect of the present invention

1. present invention demonstrates in embryo development procedure, Isl1 gene or albumen play an important role to the formation in sinus node cells whole growth cycle and function point analysis, especially for the formation of sinus node cells middle and advanced stage and function, the disappearance of Isl1 or variation can not cause embryonic death, but cause postnatal fetus to suffer from arrhythmia to be the various congenital heart diseases of cardinal symptom.

2. the present invention have also demonstrated the upstream gene as HCN4, and the regulation and control of Isl1 to HCN4 play an important role, thus the middle and advanced stage of regulation and control embryo sinus node cells is grown.

3. the present invention can be used for congenital heart disease, is especially Prenatal Screening and the diagnosis of the various congenital heart diseases of cardinal symptom with arrhythmia, is conducive to prenatal and postnatal care.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.

Embodiment 1Isl1 and HCN4 is at heart SAN cells

In order to study the effect of Isl1 in SAN formation and function, first analyze the expression of Isl1 in SAN growth course, and use several SAN cell and myocardium of atrium cell antibody (HCN4Tbx3Cx40 is purchased from abcam company) as mark.

HCN4 is pacemaker cell channel protein, starts in early days to express, in SAN differentiation and development process, play keying action at heart development.The venous sinus (sinus venosus(SV) of myocardium is expressed at E9.5, Isl1), pacemaker cell hair tonic region high expression level HCN4(Figure 1A of body early embryo).

At E10.5, Isl1 and HCN4 coexpression in embryo SAN head (Figure 1B) and afterbody (Fig. 1 C), venous valve (vv), and around the atrial myocardium (DM) (Figure 1A, B, arrow) of dorsal mesocardium.

Early stage after fetal development late period and birth, Isl1 and HCN4 coexpression is in sinus node cells (Fig. 1 D, E, I).

The expression of Isl1 in SAN reduced gradually along with the age.The pacemaker cell quantitative proportion of Isl1 and HNC4 coexpression significantly reduces after birth for 2 weeks, and reduces (Fig. 1 L) gradually along with the age increases.

Tbx3 and CX40 dyes the boundary that can mark between SAN and atrial myocardium altogether.Find at embryo E10.5 days, Tbx3 in SAN head and afterbody and venous valve and Isl1 coexpression (Fig. 1 F, G).At atrial myocardium, Isl1 is expressed in SAN(Fig. 1 H, J in Cx40 expression).

Result indicates Isl1 and grows and playing an important role in function at SAN.

Embodiment 2 reduction that Isl1 expresses in Isl1 compound sudden change mouse embryo causes the minimizing of sinus arrhythmia and SAN cell

The compound mutant mice model of the Isl1 (Isl1nLacZ/f that a 2.1 structures Isl1 not exclusively expresses ^neo+), its phenotype comparatively whole body knock-out mice is light, can survive slightly for a long time (death in E12.5 days), but comparatively Isl1 not exclusively to express mouse system phenotype heavier.

The compound mutant mice model of Isl1 uses Isl1 core LacZ to knock in mouse system (Isl-nLacZ), and Isl1 low expression mouse system (Isl1f ^neocontaining neomycin site in this mouse Isl1 intron of /+, Isl1 normal expression can be disturbed) hybridization forms.

Compound mutant mice (the Isl1nLacZ/f of result: Isl1 ^neo) not exclusively express compared with Isl1 the Isl1 that less amount is expressed by mouse system, embryo can be caused in E11.5 death (54).In tissue slice X-gal dyeing display Isl1-nLaz mouse, the expressive site of Isl1-nLaz colour developing and endogenous Isl1 is completely the same.

Consistent with shown in Fig. 1, at E9.5Isl1-nLacZ at dorsal mesocardium, around SAN and SAN, atrial endocardium expresses (Fig. 2 A, A' arrow).

SAN(Fig. 2 D, D' is expressed in) at E11.5, Isl1-nLacZ.

At the compound mutant mice of E9.5 and E11.5Isl1 at SV myocardium, SAN, the Isl1-nLacZ positive cell quantity at dorsal mesocardium position significantly reduces (Fig. 2 B, B', E, E', arrow).

Isl1 and HCN4 antibody carries out immunofluorescence dyeing and shows that Isl1 and HCN4 positive cell significantly reduces (Fig. 2 C, C') in the E9.5 days compound mutant SV of Isl1.

Conclusion: Isl1 is to SAN cell, and SV myocardial cell, propagation and the survival of the cardiac progenitor cell at heart rear portion are all necessary.For this reason, use BrdU as cell proliferation marker, use TUNEL dyeing observation of cell apoptosis.Find E9.5 embryo, SV myocardium BrdU labeled cell quantity significantly reduces, and SV right corner apoptosis quantity significantly increases (Fig. 2 G, G').

2.2 in order to study the function of the compound mutant pacemaker cell of Isl1 further, and using ultrasound cardiograph detects the changes in heart rate of mutant mice E9.5 to E11.5.Marked the position of each embryo in uterus and detect each embryo heart rate.

The compound mutant of result: Isl1 is compared obviously slack-off at the heart rate of E9.5 with the heart rate of control group E9.5, at Isl1 compound mutant E11.5 decreased heart rate more significantly (Fig. 2 H).Find that the compound mutated embryonic of Isl1 there will be intermittent cardiac extra systole, its occurrence frequency increases along with the growth of embryo simultaneously.Partial embryonic generation Prolonged Cardioplegia is had in experiment testing process.

The sudden change mouse of the early stage low expression of conclusion: Isl1 dies from irregular pulse.

In the compound mutated embryonic of embodiment 3Isl1, Isl1 expresses to reduce and causes the key gene that SNA function is relevant to express reduction

RNA in situ hybridization result shows, and HCN4, Shox2 and Tbx3 express (Fig. 3 A-C, A'-C', arrow) in the SV of control group E9.5, and in the SV of the compound mutant E9.5 of Isl1, express significantly reduction (Fig. 3 F-H, F'-H', arrow).

Cx40 and Nkx2.5 expresses at control group embryonic myocardium layer, but is not expressed in SAN (Fig. 3 D, D', E, E', arrow).In the compound mutated embryonic of Isl1, find that ectopic expression appears in Shox2 and Tbx3, and do not find that the phenomenon (Fig. 3 I, I', J, J', arrow) of ectopic expression appears in CX40 and NKX2.5.

In the compound mutated embryonic of conclusion: Isl1, Isl1 expresses reduction and can cause HCN4, and the genetic expression that the SNA functions such as Shox2 with Tbx3 are relevant reduces, and Isl1 has regulated and controled the expression of these genes, is its upstream gene.

Embodiment 4Isl1 to break up myocardial cell be still required

In the compound mutated embryonic of Isl1, broken up SAN pacemaker cell (E9.5-E11.5), in the second heart district cardiac progenitor cell and SV, SAN myocardium, the expression of Isl1 all significantly reduces.Therefore, Troponin-Cre(cTnT-Cre is used) mouse breaking up specific knockdown Isl1 in myocardial cell, breaking up the effect in sinus node cells in order to observe Isl1.

CTnT-Cre knocks out Isl1 mouse (cTnT-Cre; Isl1 mouse is) and the phenotype of Isl1 compound sudden change mouse similar, this sudden change mouse is in the embryonic stage of i.e. E9.5-E11.5 death in early days, and mutated embryonic shows the slack-off and premature beat of obvious heart rate, sees Fig. 7.

Conclusion: experiment shows, even if myocardial cell is ripe, it is required that the pacing function of expression to myocardial cell of Isl1 is still.

Embodiment 5 uses HCN4-CreERT2 knock-out mice SAN cell ISl1 can cause embryo E11.5 sinus arrhythmia at E9.5, and sinus node cells reduces and death during embryonic period

Because Isl1 continuous expression and expression time in SAN will be longer than cTnT-Cre; Therefore need to use additive method research to grow the effect of Isl1 in late period in SAN.

5.1 in E9.5 days, the SV main pacemaker areas as heart, and SAN formed distinguishable weave construction as far back as E11.5 days, the ripe also functionating at E13.5 days.Therefore main research is the above-described SAN etap.Induced by tamoxifen, HCN4-Cre can express in conducting system of heart.Use HCN4-CreERT2 and the Isl1f/f mouse hybrid with Rosa26-LacZ or YFP background, to follow the trail of the expressive site of Cre.Derivable HCN4Cre mouse is adopted to be that (HCN4-CreERT2) builds the gene knock-out mice model of specific Isl1 in SAN pacemaker cell.

Result: detect embryo heart rate with ultrasound cardiograph by every day after giving tamoxifen at E9.5 days.Find, compare with control group, HCN4-CreERT2; Isl1 sudden change mouse heart rate is significantly slack-off at E10.5 days.HCN4-CreERT2; Isl1 mutant heart rate reduces gradually along with growth course, stops jumping for a long time, most HCN4-CreERT2 at E11.5 days generation hearts; Isl1 mutant embryos is at death in E11.5 days (Fig. 4 A).

5.2 in order to detect SAN cell at HCN4-CreERT2; Distribution situation in Isl1, uses Rosa-LacZ dyeing to follow the tracks of SAN cell development and distribution situation.After embryo E9.5 gives tamoxifen, get embryo in E10.5-E11 and E11.5 and carry out X-gal dyeing (Fig. 4 B-F).

Result: control group E10.5 and 11.5 embryos, the X-gal positive cell that HCN4-CreERT2 marks is present in SV and SAN (Fig. 4 B, E, red arrow, b1, b2), this consistent with the expressive site of endogenous HCN4mRNA (Fig. 3 A).But at E10.5-E11 mutated embryonic, part X-gal positive cell disappearance (Fig. 4 E, F) in SV region.Similar with control group in E11.5, X-gal+ positive cell distributed areas, but in SV, HCN4-CreERT2 positive cell quantity significantly reduces (Fig. 4 B, C, red arrow, b1, b2, c1, c2, D).

5.3 application immunofluorescence dyeings detect the expression of E11 days Isl1 before SAN cell loss.

Result: significantly reduce the expression of Isl1 in SV and SAN (Fig. 4 G, H) in the induction of E9.5 days tamoxifens, and Isl1 has no change in pharyngeal and expression that is dorsal mesocardium region simultaneously.

HCN4-CreERT2; In SV and the SAN region of Isl1 mutated embryonic, the expression of HCN4 and Tbx3 then significantly reduces (Fig. 4 I-L), and this shows that Isl1 has regulated and controled the expression of HCN4 and Tbx3 simultaneously.

In addition, experiment proves, in the SAN region that HCN4 expresses, the cell quantity of TUNEL mark significantly increases (Fig. 8), and the cell quantity of BrdU mark reduces.

Conclusion: Isl1 grows in the survival of SAN cell and breeding and has early interimly played vital role.

Embodiment 6 uses HCN4-CreERT2 to knock out Isl1 in E11.5 embryo SAN cell can cause sinus arrhythmia, reduces the propagation of SAN cell.

Growing late period and the effect in its pacing function in order to study Isl1 at SAN, growing at SAN and having knocked out Isl1 late period.

6.1 gave tamoxifen at E11.5 days, significantly slowed down (Fig. 5 A) in the super detection of the heart of Isl1 mutated embryonic heart rate after 24 and 72 hours.But with embryo, to knock out Isl1 in early days different, and after knocking out Isl1 late period, most mutated embryonic still can be survived.

Gave tamoxifen at E13.5 days, find that mutated embryonic heart rate significantly reduces (Fig. 5 B) after 48 hrs.Research also finds that control group embryo heart rate increases gradually in Development Mouse Embryo, but HCN4-CreERT2; Isl1 mutated embryonic heart rate has no increase, and it is irregular to show changes in heart rate.(Fig. 5 B).

E14.5 days heart (induction of E11.5 tamoxifen) X-gal and galactosidase immunofluorescences that are overall and section are dyeed display altogether, X-gal positive cell quantity (Fig. 5 D, D', H) comparatively control group (Fig. 5 C in Isl1 mutated embryonic SAN, C', G) slightly reduce.Quantitative analysis display (each SAN counts 10 sections), in the SAN of Isl mutated embryonic quantity (175.5 ± 11.7/ section) (Fig. 5 D') of X-gal positive cell comparatively control group (205.6 ± 17.6/ cut into slices) (Fig. 5 C') decrease 15%.

After conclusion: SAN grows and knocks out Isl1 late period, most of embryo can be survived, but still can cause serious irregular pulse.

HCN4-CreERT2 in 6.2Isl1 mutated embryonic SAN; The proliferative cell (BrdU+) of Rosa-YFP positive cell obviously reduces.Contrary, early stage in growth, HCN4-CreERT2; Isl1 mutated embryonic SAN apoptosis has no and increases.

HCN4-CreERT2; The expression of the display of SAN section immunofluorescence dyeing result Isl1, HCN4 and the Tbx3 of Isl1 sudden change mouse significantly reduces (Fig. 5 E-H).Although but the expression of Tbx3 reduces, and in no evidence display Isl1 mutant SAN, there is the ectopic expression of NKX2.5.

The minimizing of conclusion: Isl1 causes the minimizing of multiple gene (as HCN4), and therefore, Isl1 may serve important upstream regulating and controlling effect in conducting system of heart.

Embodiment 7Isl1 has regulated and controled the key transcription factor that SAN grows and function is necessary, ionic channel, and cell cycle, the expression of cell survival gene.

In order to study the downstream target gene that the phenotypic Isl1 of aforementioned Isl1 sudden change mouse may be caused potential, use qRT-PCR to E11.5 days HCN4-CreERT2; Isl1 sudden change mouse and control group SAN cell have carried out analyzing (tamoxifen is induced at E9.5) (Fig. 6 A-C).Relevant to Isl1 or relevant with Isl1 phenotype gene is selected to analyze.

7.1 experiments find, a series of signal pathway gene down-regulated expression, comprises cell proliferation (Ccnd1, Ccnd2, Ccne1, Ccnb1, Plk1, Aurka and Cdkn1), cell survival (Bcl2) (Fig. 6 A) and heart and SAN grow necessary transcription factor (Shox2, Isl1, Mef2c, Tbx3) (Fig. 6 B).In these genes, Ccnd1 (encoding cyclin D1) and Mef2c has been proved to be the direct downstream target gene of Isl1.Meanwhile, the down-regulated expression (Atp1b1, Atp1b2, Atp2a1, Kcne1, Kcna5 and Cacna1h) of a series of ion channel gene relevant with conducting system function is also had.Gja5 and Gjd3(encodes Cx40 and Cx30.2 respectively) expression also obviously reduce.Gja1 and Gja4(encodes Cx43 and Cx37 respectively) expression then have and increase (Fig. 6 C).In addition, also find that the expression of the gene M yh6 relevant with mankind's sick sinus syndrome in Isl1 mutated embryonic SAN reduces (Fig. 6 C).

Key factor HCN4 and Tbx3 in 7.2SAN growth course is at HCN4-CreERT2; Expression in Isl1 mutated embryonic SAN significantly reduces.In order to study the direct downstream target gene whether these 2 genes are Isl1, the mouse SAN cell that suddenlys change of E14.5 to E17.5HCN4-nEGFP after FACS purifying is carried out nano-ChIP analysis (60) (51).

QPCR analyzes the gene expression abundance of different DNA in ChIP result.In order to identify that Isl1 is at evolution conservative region (ECRs) possible binding site (YTAATR), checked the region of mouse HCN4 and Tbx3 genomic upstream and each 5kb in downstream.

Result: 2 ECRs may include Isl1 binding site (Fig. 6 D, F).Tbx3-1 (chr5:119671762-119672016) is positioned at the 5 ' non-coding region of Tbx3, includes 2 adjacent TAAT sites at transcription initiation site (TSS) downstream 1271bp and 1270bp.Tbx3-2 (chr5:119684628-119685046) holds in the ECR of adjacent 465bp at a Tbx33 ', containing multiple potential Isl1 binding site.

ChIP-PCR have also discovered 2 high abundance site locus (Fig. 6 E, F) at HCN4.HCN4-2(chr9:58842538-58842979) be positioned in a 442bp intron ECR, this site has been proved to be as being an effective enhanser, and there is multiple Isl1 binding site in this region.HCN4-1(chr9:58820136-58820197) be positioned at a non-conservative region, be positioned at 3376bp place, HCN4 transcription initiation site upstream, include a potential Isl1 binding site.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. a LIM homologous structure domain transcription factor (LIM homeodomain transcription factor, Islet1, Isl1) purposes of gene or its albumen, is characterized in that, for the preparation of the reagent or the test kit that detect or predict sinus node (SAN) abnormal relative disease.

2. purposes as claimed in claim 1, it is characterized in that, described Isl1 gene or albumen come from Mammals, more preferably derive from rodent (as mouse, rat) or primate (as people).

3. purposes as claimed in claim 1, is characterized in that, the abnormal relative disease of described sinus node comprises: sick sinus syndrome or conducting system of heart are correlated with irregular pulse.

4. purposes as claimed in claim 1, it is characterized in that, described reagent comprises Isl1 Auele Specific Primer, specific antibody, probe and/or chip.

5. purposes as claimed in claim 1, is characterized in that, described detection measures amniotic fluid samples, tissue sample, blood sample, serum sample or humoral sample.

6. purposes as claimed in claim 5, it is characterized in that, described amniotic fluid samples is from mouse E8-E14 or the mankind embryo of 12 weeks 20 weeks.

7. purposes as claimed in claim 1, is characterized in that, described reagent comprises:

A () detects the nucleic acid chip of Isl1;

The specific antibody of (b) anti-Isl1 albumen; And/or

The genomic dna of (c) specific amplification Isl1, the Auele Specific Primer of mRNA or cDNA.

8. for detecting or predict a test kit for the abnormal relative disease of sinus node, it is characterized in that, described test kit comprises and detects the nucleic acid chip of Isl1 or the Auele Specific Primer of specific amplification Isl1 nucleotide sequence and specification sheets; Wherein said specification sheets comprises following content: detected by the expression and/or activity that measure Isl1 gene or albumen or predicted sinus node (SAN) abnormal relative disease.

9. test kit as claimed in claim 8, it is characterized in that, following content also recorded by described specification sheets: detect the required Isl1 gene of object or the expression of albumen and/or activity, if significantly lower than normal population, then illustrate, this detected object suffers from the probability of the abnormal relative disease of sinus node higher than normal population.

10. prepare a method for animal model, it is characterized in that, comprise step:

Genotypic for Isl1hypo/+ rodent and the genotypic rodent of Isl1+/nLacZ are carried out mating or genotypic for Isl1hypo/+ rodent and the genotypic rodent of Isl1hypo/+ are carried out mating, the rodent filial generation thus the acquisition low expression of Isl1 compound (Isl1hypo/nLacZ) makes a variation.