CN1884522A - Method for preparing single blastomere chromosome - Google Patents
Method for preparing single blastomere chromosome Download PDFInfo
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- CN1884522A CN1884522A CN 200610052319 CN200610052319A CN1884522A CN 1884522 A CN1884522 A CN 1884522A CN 200610052319 CN200610052319 CN 200610052319 CN 200610052319 A CN200610052319 A CN 200610052319A CN 1884522 A CN1884522 A CN 1884522A
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Abstract
The invention provides a method for preparing individual blastomere chromosome, and the said method is as follows: removing germ hill from normal fertilized ovum in prokaryotic period and performing enucleation to obtain cell plastid, in addition selecting individual human blastomere by biopsy, performing electric anabranch to the said blastomere and the cell plastid to obtain heterocaryon, introducing the prematured chromosome to conglomerate to shape with the obtained heterocaryon, then performing low permeability to the introduced heterocaryon, afterwards fixing and colouring the heterocaryon, finally obtaining the said individual blastomere chromosome. The invention obtains the method for preparing individual blastomere chromosome with high efficiency and fine quality by further improving and perfecting the electric anabranch solution, chromosome evoked solution, low permeability solution and mounting step, the prepared chromosome is of intelligible shape and of high quality, the method connected with G-bandings or chromosome paint-on and probe FISH technology can overcome the shortage existed in present interphase nucleus FISH technology and can make the PGD diagnostic range extended to the whole chromosome complement.
Description
(1) technical field
The present invention relates to a kind of method for preparing single blastomere chromosome, belong to chromosomal disorder PGD field.
(2) background technology
Chromosomal disorder is the serious inherited disease of a big class, and severe patient is died young in early days and caused spontaneous abortion the embryo, even a few symptoms is also often unusual with congenital multiple deformity and growth, intelligence, sexual abnormality or fertility than the lighter's survival birth.Chromosomal disorder is very harmful to the mankind, but does not have the treatment good plan, is mainly prevented by genetic counseling and antenatal diagnosis at present.But in a single day genetic counseling lack the specific aim means, and antenatal diagnosis is traumatic means only based on health propaganda and education, and makes a definite diagnosis, need take unwillingly property miscarriage termination of pregnancy, brings the body and mind misery to mother, also relates to the dispute of ethics aspect simultaneously.PGD technology (Preimplantation genetic diagnosis, PGD) be under the situation that does not influence the fetal development of implanting parent, a blastomere cell drawing this embryo carries out genetic diagnosis, again according to diagnostic result rejecting abnormalities embryo, select the embryo transfer of no heredity problem to go back to the parent uterus and be born the normal-sub technology in generation, in theory can be from the generation of control of heredity disease on the source, therefore become a kind of new inborn defect interference method and received publicity day by day.
The PGD of chromosomal disorder mainly depends on interphasic nucleus fluorescence in situ hybridization technique (FISH) at present, but this technology only can provide limited chromosomal region domain information, can not detect all chromosome abnormalty types, therefore there is certain defective aspect the susceptibility of diagnosing, the accuracy, and restricting the conventional means that this technology becomes the control of inborn defect source.Obtain the metacinesis phase of embryonic cell, carry out the whole chromosome group analysis of embryonic cell, can overcome the limitation of interphasic nucleus FISH, can detect all chromosome abnormalty types in theory.
But prepare the chromosomal inefficiency of single embryonic cell with conventional cytogenetic methods, the division of acquisition is also analyzed because of quality lowly is difficult to carry out follow-up PGD mutually.Nuclear transplantation shows, exist a kind of endochylema factor of controlling the foreign nucleus behavior in the animal ovum, move into the ovum of stoning when the nucleus that is in mitotic division each period after, a series of metamorphosis (promptly nuclear is modified) all takes place in foreign nucleus, comprise the nuclear membrane disintegration that moves into nuclear, chromatin condensation and premature chromosome condensation (Prematurechromosome condensation, process such as PCC).
(3) summary of the invention
The present invention promptly is for the preparation method of a kind of gained karyomit(e) efficient height, the measured single blastomere chromosome of matter is provided.
In order to reach goal of the invention the technical solution used in the present invention be:
A kind of method for preparing single blastomere chromosome, described method is as follows: normal protokaryon phase zygote is removed ovarian cumulus, carried out the stoning operation, obtain cytosome, single people's blastomere that alternative is learnt from else's experience biopsy carries out the electricity fusion with described blastomere and cytosome, obtains heterokaryon, institute's electricity consumption fusion liquid composition final concentration is in the described electric fusion steps: sorbyl alcohol 0.28M, sal epsom 0.1mM, bovine serum albumin (BSA) 0.3%, surplus is a water; Place the HOF who contains Omaine 0.05～0.1 μ g/mL to cultivate 6～13 hours the gained heterokaryon, handle with the HOF who contains 0.1～0.2 μ g/mL cytochalasin D, 1.5～2.0 μ M calyculins again, induce premature chromosome condensation to form, heterokaryon after will inducing again places hypotonic 15～20 minutes of the 0.1% Trisodium Citrate BP solution that contains 2% bovine serum albumin, then heterokaryon is fixed and dyeed, can obtain described single blastomere chromosome.Described dyeing is Giemsa staining or DAPI dyeing, and used percentage concentration is mass percent concentration among the present invention.Described cytosome promptly is the cytoplasmic receptor of people's blastomere chromosome, uses mouse protokaryon phase zygote as cytoplasmic receptor usually, and protokaryon phase ovum that also can end user's abnormal fertilization is as cytoplasmic receptor.
Electricity merges in the liquid selects for use N.F,USP MANNITOL, sorbyl alcohol, sucrose, glucose or maltose as conditioning agent usually, play the effect that steady seepage is pressed, find after further study, merge with the electricity of N.F,USP MANNITOL configuration that liquid easily forms white crystals and surface tension is big, ovocyte easily is attached to the bottom therein and is difficult to pressure-vaccum, lose easily in moving the ovum process, the electricity fusion liquid that disposes with sorbyl alcohol does not then have this phenomenon.In addition, in 0.1% Trisodium Citrate BP solution, add BSA and can play the effect that prevents the heterokaryon membranolysis, and can make the heterokaryon that removes zona pellucida be not easy to be bonded at container bottom, heterokaryon is not lost in operation, so more helps improving chromosomal efficient of acquisition and quality.
Described heterokaryotic fixing step is as follows: earlier heterokaryon was pre-fixed in stationary liquid I 3～5 minutes, again heterokaryon is transferred to slide and drips 1 stationary liquid II and fix 3～5 minutes, be transferred at last among the stationary liquid III and fix 1 minute, described stationary liquid I is methyl alcohol, acetate, 5: 1: 4 mixed solution of water volume ratio, described stationary liquid II is methyl alcohol, 3: 1 mixed solution of acetate volume ratio, and described stationary liquid III is methyl alcohol, acetate, 3: 3: 1 mixed solution of water volume ratio.Usually used heterokaryotic to be fixed as for two steps fixing, adopts three step fixation methods among the present invention, fixing by the 3rd step, can make fixed heterokaryon background more clear, and it is desirable that karyomit(e) is disperseed.Like this, make fixing means relatively gentle, strong operability, the karyomit(e) background of preparation is clear, both can avoid disperseing bad phenomenon because of the karyomit(e) that tenuigenin digestion not exclusively causes, can reduce the chromosome dyad that causes because of excessive dispersion again and lose phenomenon.
Electricity is fused in the described electric fusion steps: single pulsed voltage 0.8～0.9KV/cm, effect 10～20 microseconds.
Be example with the mouse in the described protokaryon phase zygote experimentation, obtain by following steps: female mice abdominal injection pregnant mare serum 7.5～10 international unit of giving normal 3～5 ages in week/only, injection chorionic-gonadotropin hormone 7.5～10 international unit after 48 hours/only, mate with the male mouse that normal reproductive performance is arranged, put to death female mouse after 24～26 hours at the injection chorionic-gonadotropin hormone, isolate uterine tube, HOF with bovine serum albumin mass content 10% goes out protokaryon phase zygote, and remove the zygote ovarian cumulus with the hyaluronic acid of 80IU/mL, described protokaryon phase zygote.
Described cytosome is obtained by following steps: after placing the HOF who contains final concentration 0.2～0.4M sucrose, 0.8～1.0 μ g/mL cytochalasin D, 0.1～0.3 μ g/mL Omaine to handle 5～10 minutes protokaryon phase zygote, utilize microscope work to carry out stoning again, obtain cytosome.
Concrete, described method steps is as follows:
(1) female mice abdominal injection pregnant mare serum 7.5～10 international unit of giving normal 3～5 ages in week/only, injection chorionic-gonadotropin hormone 7.5～10 international unit after 48 hours/only, mate with the male mouse that normal reproductive performance is arranged, put to death female mouse after 24～26 hours at the injection chorionic-gonadotropin hormone, isolate uterine tube, go out protokaryon phase zygote with the HOF who contains bovine serum albumin 10%, and remove the zygote ovarian cumulus, get described protokaryon phase zygote with the hyaluronic acid of 80IU/mL;
(2) step (1) gained protokaryon phase zygote is placed to contain final concentration be after the HOF of 0.2～0.4M sucrose, 0.8～1.0 μ g/mL cytochalasin D, 0.1～0.3 μ g/mL Omaine handles 5～10 minutes, utilize microscope work to carry out stoning again, obtain cytosome;
(3) choose single people's blastomere, blastomere and cytosome are moved into contain among the HOF of lectins 300 μ g/mL, cytosome, blastomere are arranged, make paired gathering through biopsy;
(4) paired cytosome, blastomere immigration are contained final concentration sorbyl alcohol 0.28M, sal epsom 0.1mM, the electricity of bovine serum albumin 0.3% merges the liquid balance, carries out electricity again and merges, and gets heterokaryon; Electricity fusion program is: single pulsed voltage 0.8～0.9KV/cm, effect 10～20 microseconds;
(5) the gained heterokaryon is placed the HOF who contains Omaine 0.05～0.1 μ g/mL cultivated 6～13 hours, handle with the HOF who contains 0.1～0.2 μ g/mL cytochalasin D, 1.5～2.0 μ M calyculins again, induce premature chromosome condensation to form; Usually adopt okadaic acid to carry out inducing of premature chromosome condensation, have and discover that calyculin is 20 times of okadaic acid in the precocious chromosomal efficient of inducing of lymphocyte, the present invention adopts 1.5～2.0 μ M calyx sponges to induce the precocious karyomit(e) of blastomere, finds to have the phenomenon of improving of the efficient of inducing.
(6) heterokaryon after will inducing again places hypotonic 15～20 minutes of the 0.1% Trisodium Citrate BP solution that contains 2% bovine serum albumin, earlier heterokaryon was pre-fixed in stationary liquid I 3～5 minutes then, after treating that heterokaryon tenuigenin is transparent, heterokaryon is transferred to slide and drips 1 stationary liquid II, treat that stationary liquid II is almost dry, drip 2～3 of stationary liquid II when heterokaryon manifests, at last slide is transferred to stationary liquid III and fixes 1 minute, after the slide drying, use 10% Giemsa staining again 5 minutes, and can obtain described single blastomere chromosome; Described stationary liquid I is methyl alcohol, acetate, 5: 1: 4 mixed solution of water volume ratio, and described stationary liquid II is methyl alcohol, 3: 1 mixed solution of acetate volume ratio, and described stationary liquid III is methyl alcohol, acetate, 3: 3: 1 mixed solution of water volume ratio.
The beneficial effect for preparing the method for single blastomere chromosome of the present invention is mainly reflected in: tuck in by electricity is merged, karyomit(e) induced liquid, hypotonic medium, and the further improvement of fixing step and perfect, obtained the preparation method of efficient height, matter measured single blastomere chromosome, make that chromosome morphology is clear, quality is high, show the FISH technology that band or whole chromosome are smeared probe in conjunction with G-, can overcome the deficiency that existing interphasic nucleus FISH technology exists, make the diagnostic area of PGD be extended to whole genome.
(4) Figure of description
Fig. 1 is the painted single people's blastomere chromosome of DAPI;
Fig. 2 is single people's blastomere chromosome of Giemsa staining;
Fig. 3 is the karyotyping figure of single people's blastomere chromosome;
Fig. 4 is the karyotyping figure of single people's blastomere chromosome;
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the acquisition of people's blastomere chromosome during as cytoplasmic receptor with mouse protokaryon phase zygote
Inject the pregnant mare serum of 9.0 international unit for earlier the female mice in 4 ages in week, injection 9.0 international unit chorionic-gonadotropin hormones (HCG) mate with the male mouse that reproductive performance is arranged after 48 hours.Put to death female mouse after 24 hours in HCG injection, isolate uterine tube, go out protokaryon phase zygote, and remove the ovarian cumulus of zygote with the Unidasa of 80IU/ml with the HOF (mHTF) who contains 10% bovine serum albumin (BSA).
Zygote places the mHTF that contains 0.3M sucrose, 0.9 μ g/mL cytochalasin D and 0.2 μ g/mL Omaine to handle after 8 minutes earlier, utilizes micromanipulation system to carry out the stoning of protokaryon phase zygote again.Ovum is fixed with the ovum pin of holding of internal diameter 120 μ m, and makes a call to a hole with laser-light transparent band punching instrument at 3 o ' clock positions, uses the angle pin stoning of internal diameter 15 μ m again.The zygote of stoning is dissolved zona pellucida with 0.3% PRONASE A, removes polar body with the pasteur pipet of drawing-down, obtains cytosome.
Select the 3rd day and divide to the embryo to be measured of 6-8 cell stage and carry out the blastomere biopsy.The embryo utilizes micromanipulation system to carry out zona pellucida perforate and blastomere biopsy after hatching 5 minutes earlier in the nutrient solution of no calcium magnesium.The embryo fixes with the ovum pin of holding of internal diameter 120 μ m, and in 3 o ' clock position perforates, flat mouthful with internal diameter 35 μ m takes out a blastomere again with laser-light transparent band punching instrument, and the embryo after the biopsy proceeds to cultivate.
The cytosome immigration of blastomere and stoning is contained among the mHTF of 300 μ g/mL lectinses (PHA), cytosome, blastomere are arranged, both are assembled in pairs with the pasteur pipet of drawing-down.
Blastomere-cytosome is merged in the liquid moving into electricity, and electricity merges consisting of of liquid: sorbyl alcohol 0.28M, sal epsom 0.1mM, bovine serum albumin mass concentration 0.3%.Blastomere-cytosome is moments later carried out electric mixing operation at ECM 830 cytogamy electronic operation instrument to balance in electricity fusion liquid, and the program that electricity merges is: single pulsed voltage 0.8～0.9KV/cm, effect 10-20 microsecond.
Merge the heterokaryon that forms and in the HOF (HTF) who contains 0.8 μ g/mL Omaine, cultivated 10 hours, handled 1 hour the formation of inducing PCC again with the HTF that contains 1.8 μ M calyculins and 0.2 μ g/mL cytochalasin D.
Hypotonic 20 minutes of the 0.1% Trisodium Citrate BP solution of 2%BSA will be induced the heterokaryon that finishes to place to contain, at stationary liquid I (methyl alcohol: acetate: pre-fixed 3 minutes water (volume ratio)=5: 1: 4), after treating heterokaryotic tenuigenin bleach, again heterokaryon is transferred to slide and drips 1 stationary liquid II (methyl alcohol: acetate (volume ratio)=3: 1), treat that stationary liquid II is almost dry, when manifesting, heterokaryon drips 3 stationary liquid II again, at last slide is transferred to stationary liquid III (methyl alcohol: acetate: fix 1 minute water (volume ratio)=3: 3: 1), treat that slide is in the humidity incubator slowly after the drying, use the Giemsa staining 5 minutes of mass concentration 10% again, promptly obtain blastomere chromosome of the present invention.
Embodiment 2: the acquisition of single people's blastomere chromosome during as cytoplasmic receptor with people's protokaryon phase zygote of abnormal fertilization
Collect people's abnormal fertilization ovum that human assisted reproduction laboratory abandons, comprise many protokaryons ovum that single protokaryon and three protokaryons are above, carry out the stoning operation after, utilization embodiment 1 described method prepares single people's blastomere chromosome.Utilization DAPI dyeing (DAPI content 0.5 μ g/mL, dyeed 5 minutes) obtain single people's blastomere chromosome and see Fig. 1, utilization Ji Samu dyeing (5% Ji Samu dyeing 5 minutes) obtains single people's blastomere chromosome and sees Fig. 2, obtains single people's blastomere chromosome caryogram distribution plan and sees Fig. 3, Fig. 4.
By Fig. 1～4 as can be seen, utilization the inventive method, single people's blastomere chromosome form of gained is very clear, quality is higher, can smear the whole chromosome composition of the FISH technology for detection pre-implantation embryos of probe by apparent band of G-and whole chromosome, can be applied among the PGD of various numerical abnormalities, textural anomaly chromosomal disorder, can on the source, control the generation of chromosomal disorder, have important prenatal and postnatal care meaning.
Claims (6)
1. method for preparing single blastomere chromosome, it is characterized in that described method is as follows: normal protokaryon phase zygote is removed ovarian cumulus, carried out the stoning operation, obtain cytosome, single people's blastomere that alternative is learnt from else's experience biopsy carries out the electricity fusion with described blastomere and cytosome, obtains heterokaryon, institute's electricity consumption fusion liquid composition final concentration is in the described electric fusion steps: sorbyl alcohol 0.28M, sal epsom 0.1mM, bovine serum albumin 0.3%, surplus is a water; Place the HOF who contains Omaine 0.05～0.1 μ g/mL to cultivate 6～13 hours the gained heterokaryon, handle with the HOF who contains 0.1～0.2 μ g/mL cytochalasin D, 1.5～2.0 μ M calyculins again, induce premature chromosome condensation to form, heterokaryon after will inducing again places hypotonic 15～20 minutes of the 0.1% Trisodium Citrate BP solution that contains 2% bovine serum albumin, then heterokaryon is fixed and dyeed, can obtain described single blastomere chromosome.
2. the method for preparing single blastomere chromosome as claimed in claim 1, it is characterized in that described heterokaryotic fixing step is as follows: earlier heterokaryon was pre-fixed 3～5 minutes in stationary liquid I, again heterokaryon is transferred to slide and drips 1 stationary liquid II and fix 3～5 minutes, be transferred at last among the stationary liquid III and fix 1 minute, described stationary liquid I is a methyl alcohol, acetate, 5: 1: 4 mixed solution of water volume ratio, described stationary liquid II is a methyl alcohol, 3: 1 mixed solution of acetate volume ratio, described stationary liquid III is a methyl alcohol, acetate, 3: 3: 1 mixed solution of water volume ratio.
3. the method for preparing single blastomere chromosome as claimed in claim 1 or 2 is characterized in that electricity is fused in the described electric fusion steps: single pulsed voltage 0.8～0.9KV/cm, effect 10～20 microseconds.
4. the method for preparing single blastomere chromosome as claimed in claim 1 or 2, it is characterized in that described protokaryon phase zygote is obtained by following steps: female mice abdominal injection pregnant mare serum 7.5～10 international unit of giving normal 3～5 ages in week/only, injection chorionic-gonadotropin hormone 7.5～10 international unit after 48 hours/only, mate with the male mouse that normal reproductive performance is arranged, put to death female mouse after 24～26 hours at the injection chorionic-gonadotropin hormone, isolate uterine tube, HOF with bovine serum albumin mass content 10% goes out protokaryon phase zygote, and remove the zygote ovarian cumulus with the hyaluronic acid of 80IU/mL, described protokaryon phase zygote.
5. the method for preparing single blastomere chromosome as claimed in claim 1 or 2, it is characterized in that described cytosome is obtained by following steps: after placing the HOF who contains final concentration 0.2～0.4M sucrose, 0.8～1.0 μ g/mL cytochalasin D, 0.1～0.3 μ g/mL Omaine to handle 5～10 minutes protokaryon phase zygote, utilize microscope work to carry out stoning again, obtain cytosome.
6. the method for preparing single blastomere chromosome as claimed in claim 1 is characterized in that described method steps is as follows:
(1) female mice abdominal injection pregnant mare serum 7.5～10 international unit of giving normal 3～5 ages in week/only, injection chorionic-gonadotropin hormone 7.5～10 international unit after 48 hours/only, mate with the male mouse that normal reproductive performance is arranged, put to death female mouse after 24～26 hours at the injection chorionic-gonadotropin hormone, isolate uterine tube, go out protokaryon phase zygote with the HOF who contains bovine serum albumin 10%, and remove the zygote ovarian cumulus, get described protokaryon phase zygote with the hyaluronic acid of 80IU/mL;
(2) step (1) gained protokaryon phase zygote is placed to contain final concentration be after the HOF of 0.2～0.4M sucrose, 0.8～1.0 μ g/mL cytochalasin D, 0.1～0.3 μ g/mL Omaine handles 5～10 minutes, utilize microscope work to carry out stoning again, obtain cytosome;
(3) choose single people's blastomere, blastomere and cytosome are moved into contain among the HOF of lectins 300 μ g/mL, cytosome, blastomere are arranged, make paired gathering through biopsy;
(4) paired cytosome, blastomere immigration are contained final concentration sorbyl alcohol 0.28M, sal epsom 0.1mM, the electricity of bovine serum albumin 0.3% merges the liquid balance, carries out electricity again and merges, and gets heterokaryon; Electricity fusion program is: single pulsed voltage 0.8～0.9KV/cm, effect 10～20 microseconds;
(5) the gained heterokaryon is placed the HOF who contains Omaine 0.05～0.1 μ g/mL cultivated 6～13 hours, handle with the HOF who contains 0.1～0.2 μ g/mL cytochalasin D, 1.5～2.0 μ M calyculins again, induce premature chromosome condensation to form;
(6) heterokaryon after will inducing again places hypotonic 15～20 minutes of the 0.1% Trisodium Citrate BP solution that contains 2% bovine serum albumin, earlier heterokaryon was pre-fixed in stationary liquid I 3～5 minutes then, after treating that heterokaryon tenuigenin is transparent, heterokaryon is transferred to slide and drips 1 stationary liquid II, treat that stationary liquid II is almost dry, drip stationary liquid II2 when heterokaryon manifests～3, at last slide is transferred to stationary liquid III and fixes 1 minute, after the slide drying, use 10% Giemsa staining again 5 minutes, and can obtain described single blastomere chromosome; Described stationary liquid I is methyl alcohol, acetate, 5: 1: 4 mixed solution of water volume ratio, and described stationary liquid II is methyl alcohol, 3: 1 mixed solution of acetate volume ratio, and described stationary liquid III is methyl alcohol, acetate, 3: 3: 1 mixed solution of water volume ratio.
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