CN1217746A - Pluripotent rabbit embryonic stem (ES) cell lines and their use in generation of chimeric rabbits - Google Patents

Pluripotent rabbit embryonic stem (ES) cell lines and their use in generation of chimeric rabbits Download PDF

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CN1217746A
CN1217746A CN97194183A CN97194183A CN1217746A CN 1217746 A CN1217746 A CN 1217746A CN 97194183 A CN97194183 A CN 97194183A CN 97194183 A CN97194183 A CN 97194183A CN 1217746 A CN1217746 A CN 1217746A
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R·默里迪斯
L·斯库恩詹斯
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Katholieke Universiteit Leuven
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Abstract

The present invention relates to a rabbit embryonic stem (ES) cell line, comprising at least 70 %, preferably 80 to 90 % undifferentiated cells and obtainable by isolating the inner cell mass of 5.5 days postcoitus blastocysts and culturing them on feeder cells in rabbit ES medium. The invention further relates to further optimization of derivation and maintenance of the cell line and to the use thereof in inter alia generation of chimeric rabbits.

Description

Pluripotent rabbit embryonic is done (ES) clone and in the aborning application of chimeric rabbits
The present invention relates to that new rabbit embryonic is done (ES) clone and in the aborning application of chimeric rabbits.
Gene targeting (by do homologous recombination in (ES) cell the embryo) technology allows with required and mode operator gene (Capecchi, 1989 regulation; Robertson, 1987; Bradley, 1987).In brief, goal gene is incorporated in the plasmid transfer vector, but and by replacing its partial sequence (inactivation) with the foreign DNA of coding selective marker or changing by the gene order (target mutagenesis) of suddenling change.Then these the gene of inactivation or sudden change be imported into ES cell (each cell all has the potentiality that develop into intact animal).Then at the in-vitro screening natural gene by the clone of the heterozygote alternate ES cell of inactivation, these clones that filter out are imported in the fetal tissues of implanting again.This process produces chimaeric animals, and these animals screen the kind system that is used for the inactivation gene and transmit.Through breeding and selecting, so produced the transgenic animal of goal gene disappearance (" knocking out ").
Utilize the technology of latest developments to make wild-type or sudden change ES cell and tetraploid embryo cohesion can produce the mouse (Nagy et al., 1993) of the complete derivation of ES cell.Optionally, the ES cell can be used for the importing of genetic material by non-homogeneous reorganization, allows to carry out in the animal that lives the research of hereditary change.
The variation that transgenic technology and ES cell technology are applied to gene function provide some important human diseasess animal model (summary is referring to Wilson, 1996; Rubin and Barsch, 1996).Yet this technology only is successful in mouse at present.Although for many application, mouse is useful, has some significantly restrictions (for example size limits and can not produce Human diseases) to limit its potential and uses.Therefore what detect that phenotype result or function lose sudden change will be very valuable than the large animal model.
The pluripotency ES cell of confirming has obtained separating in many other species, comprises hamster (Doetschman etc., 1988), pig (Evans etc., 1990; Piedrahita etc., 1990; Notarianni etc., 1990; Talbot etc., 1993), sheep (Notarianni etc., 1990), ox (Evans etc.; Saito etc., 1992), mink (Sukoyan etc., 1993), rabbit (Graves and Moreadith, 1993), rat (Iannaccione etc., 1994), people (Bongso etc., 1994) and primates (Thomson etc., 1995).Again in culture, produce chimeric ES cell yet only in mouse and rat, set up after importing blastaea, and up to the present all effort in rabbit and other species are failed all.
Fresh inner cell mass (ICM) cell from the rabbit blastaea has shown generation (Gardner and Munro, 1974 that allow chimeric rabbits after injection is subjected to stomatoblastula; Moustafa, 1974; Babinet and Bordenave, 1980), Yang etc. in addition, (1993) have been reported from the ICM of fresh separated and 3 days ICM cell of vitro culture and have been produced chimeric rabbits.
Yet for allow gene targeting by homologous recombination in the ES cell, the clone that can keep the longer time and have certified generation chimaeric animals potential in cultivation is essential for the offspring of generation purpose hereditary change.Before the present invention, except mouse and rat, there is not the clone of other such animal to be separated.
The derivation of the rabbit ES cell of the pluripotency of confirming was reported (1993) by Grave and Moreadith in the past.Two kinds of main cell types have appearred later in embryo's continuous passage of shifting out.One type has the form identical with the elementary wart of trophectoderm, and cell is responsible for embryo's implantation.Second type presents typical epithelium sample, is considered to represent rabbit ES cell (Fig. 1).Although the ES cell of these affirmations can produce embryo-likebody (by representing ectoderm, mesoderm and endoblastic various kinds of cell type are formed), but they are (Grave and Moreadith, 1993 after the epithelioid cell is imported the blastaea that derives from New Zealand's white race (NZW); Grave and Moreadith, the result's (table 1) who does not deliver), perhaps nucleus is shifted into the white zygote of the New Zealand of examining after (Du etc., 1995) can not produce chimeric rabbits.This shows with these ES cells can not produce the mosaic possibility because the obstacle of planting.Do not have chimaeric animals to produce but the data of table 1 show from the epithelioid cell system of four detections, each epithelioid cell that surveys system of institute is from single inner cell mass.
Table 1: with the result (result who does not deliver, Graves and Moreadith) of the ES clone injection rabbit embryonic of determining
Clone (%) Embryo stage The # injection Birth (%) Mosaic
????GM3 ????GM4 ????GM8 ????GM4 Blastaea blastaea morula morula ????46 ????24 ????84 ????14 ????29(63) ????2(8) ????6(7) ????5(35) ????0(0) ????0(0) ????0(0) ????0(0)
Sum ????168 ????42(25) ????0(0)
Niemann also attempts to separate with Strelchenko (1994) and keeps pluripotency rabbit (California kind) ES cell, although but the ES cell (keeping for 12 generations altogether) that can observe affirmation and host's blastaea (Chinchilla, Black Rex) cohesion of inner cell mass, but there be no evidence to suggest has chimeric offspring's generation, the rabbit embryonic that the ES cell of these affirmations was collected from post-coitum in 4 days.Therefore, up to the present, all generations are stablized the effort of pluripotency rabbit ES cell (have and be injected into the potentiality that are subjected to produce behind the stomatoblastula performance of chimaeric animals) and are failed.
Therefore target of the present invention is deriving of dried (ES) cell of improvement rabbit embryonic and keeps, make it to have the potential that produces the performance of chimeric rabbits after being subjected to stomatoblastula being injected into, therefore can produce offspring's (, using for example gene or nucleus transfer) of wild-type or hereditary change by homology or non-homogeneous reorganization.
Find at present to pass through to separate the inner cell mass of 5.5 days blastaeas of post-coitum and in rabbit ES substratum their are cultivated on feeder cell, can obtain a rabbit embryonic and do (ES) clone, it contains at least 70%, preferably 80 to 90% undifferentiated cells.
Foundation rabbit ES substratum of the present invention comprises the Eagle substratum of the modification of high glucose Dulbecco, 4mM L-glutaminate, 0.1mM2-mercaptoethanol, 148 units/ml penicillin G sodium salt, 148 milligrams/ml Vetstrep, 4 milligrams/ml Sigma I8405,10 3Unit/ml mouse leukemia supressor, 20% foetal calf serum, 1.5%MEM non-essential amino acid solution.
The preferred mouse embryo fibroblasts of feeder cell, from 12.5 days mice embryonic, and with in the petri plate of every 10cm 3 to 4 * 10 6Cell density uses.
The preferred tryptic digestion method of using a kind of improvement, it comprises uses the tryptic digestion substratum, and this substratum comprises 1% collagenase, 1% chicken serum and trypsinase-EDTA of 0.03% in phosphate buffered saline buffer.Because it is slower that mouse embryo fibroblasts and trophectoderm cell separate in this substratum, so this substratum allows going down to posterity of ES cell selective.
According to cell of the present invention, by their following properties identification: the negative staining that surpasses the forming of 10 three-dimensional clones in back of going down to posterity, alkaline phosphatase positive staining and cytokeratin 18 and vimentin.
The invention further relates to the application of ES clone in producing chimeric rabbits, for example ensuingly be injected into the blastaea injection that is subjected to stomatoblastula or embryo's cohesion or consideration convey and move.
ES clone of the present invention also can be used for gene alteration or by kind being to transmit the rabbit that is used to produce gene alteration by homology or non-homogeneous reorganization.
Can cause the research of (newly) gene with differentiation or separate according to the application of clone of the present invention.
Therefore, the present invention has caused the deriving of improved pluripotency ES cell, has surpassed keeping of going down to posterity several times and in the application that successfully produces in the chimaeric animals in cultivation.
Have the deriving and keep of the rabbit ES cell of the potentiality that are injected into the performance that produces chimeric rabbits after being subjected to stomatoblastula, can to plant be the offspring of transmission target mutation with allowing to produce homology or non-homogeneous reorganization after in these pluripotencies ES cell.In addition, the pluripotency of rabbit ES cell will allow them to be divided into specific cell type, with research or separate new gene.
The present invention will obtain explanation in the following embodiments, and these examples are not to be used for limiting the scope of the invention.On basis of the present invention, some changes and improvements are to it will be readily apparent to those skilled in the art that.Embodiment 1 cell culture condition
From ES clone GM3 (according to Grave and Moreadith (1993) (Fig. 1) description from Holland's band kind of rabbit embryonic), formed with respect to Grave and the described method of Moreadith (1993), stablized the cell culture condition of the ratio of not breaking up the ES cell.After the ES injection cell is advanced the segmentation cavity of the white blastaea of New Zealand, just might produce mosaic for the first time like this, this point will prove in example 2.
The pair cell culture condition, the density of mouse embryo fibroblast (MEF) feeder layer, the age that is used for the mice embryonic of derivation MEFS carries out some and changes, and is used for the tryptic digestion substratum of isolated cell to go down to posterity.
The substratum that Grave and Moreadith (1993) use comprises the Eagle substratum of the modification of high glucose Dulbecco, 4mM L-glutaminate, 0.1mM 2 mercapto ethanol, 148 units/ml penicillin G sodium salt, 148 milligrams/ml Vetstrep.
According to the present invention, can change or increase following fill-in: 4 μ g/ml Sigma I8405s, 10 3Unit/ml mouse leukemia supressor, 20% foetal calf serum, 1.5%MEM non-essential amino acid solution.
Rabbit ES cell is (in every 10cm petri plate 1.5 to 3 * 10 6Individual cell) grow near sub-saturated stagnating on the mitotic mouse embryo fibroblasts with mitomycin, every 4-6 days with the ES passage to the feeder cell of prepared fresh (in each 10cm culture dish 3 to 4 * 10 6Individual cell).Every day is with above-mentioned improved culture medium culturing cell.Culture dish is stored in 39 ℃, contains 5%CO 2Damp atmosphere in.Mouse embryo fibroblasts is from 12.5 days mice embryonic, and is used in the first-generation.Density (each 10cm culture dish 3 to 4 * 10 that mouse embryo fibroblasts increases 6, in contrast to 2 to 3 * 10 6(Graves and Moreadith, 1993)) with the embryo who uses 12.5 days, reduced the differentiation of ES cell significantly.
It is very crucial that the reduction of differentiation proves, because have only undifferentiated cell to preserve the ability that is integrated into the recipient embryo inner cell mass, this is the precondition that produces chimeric offspring.
In addition, use the method for improved selectivity tryptic digestion to allow trophectoderm cell (can induce the differentiation of ES cell) is removed from culture.The tryptic digestion substratum comprises 0.1% collagenase, 1% chicken serum and 0.03% trypsinase-EDTA (the Gibco catalog number (Cat.No.): 25200) that is present in the phosphate buffered saline buffer.Optionally the tryptic digestion substratum allows the selectivity of ES cell to go down to posterity, because mouse embryo fibroblasts is separated slowlyer than ES cell from culture dish with the trophectoderm cell.Embodiment 2 chimeric generations
By Graves and Moreadith (1993) derivation from Holland band kind of embryo, and be kept at GM3 cell under the culture condition among the embodiment 1, be used to produce chimaera progeny as described below.The epithelium sample clone's of the ES cell of confirming blastaea injection after being injected into rabbit embryonic, also never produced chimeric rabbits (table 1) in the past.In Shuo Ming the further experiment, the GM3 cell that derived from for 12 generations is stored under the improved culture condition below, this conditional stability the alkaline phosphatase enzyme positive, the ratio of undifferentiated ES cell.
Sexually matured New Zealand white race doe stimulates hormone (FSH-0.4 with continuous 6 the subcutaneous injection pig follicles in 12 hours interval, 0.4,0.5,0.5,0.5,0.5mg), and the human chorionic gonadotrophin (h CG) of the 10 hours intravenous injection 75 IU ovulation that exceeds the speed limit behind the last dosage of FSH.After the hCG injection, the male rabbit mating of doe and maturation of the same race.
Post-coitum 90 hours by being used in 39 ℃, contains 5%CO 2Damp atmosphere in added 3% bovine serum albumin (Cohn component V) and the 5% microbiotic/antimitotic solution of pre-equilibration (the phosphate buffered saline buffer flushing uterine cavity of Dulbecco NY) is to reclaim blastaea from horn of uterus for Gibco, Grand Island.
Go in the segmentation cavity of New Zealand's white race blastaea to produce mosaic (table 2) by injecting epithelium sample alkaline phosphatase alkaline phosphatase male ES cell negative and three dimensional growth.Use 20 to 300 injection cells to add up to 287 New Zealand's white race blastaeas from GM3 clone.Under the Zeiss inverted microscope, under the 250x magnification, carry out the blastaea injection with the GM3 cell with the difference interference light.Carry out micrurgy with daily use in the Narashige of mouse micromanipulator.
To implant the proximal part of acceptor New Zealand each horn of uterus of white race with 5 to 10 embryos of GM3 injection cell again with little otch and sharp glass pipette.This acceptor has been injected hCG has accepted 75 IU after 14 hours hCG in donor.With under the situation of ketamine/Xylazin mixture general anesthesia, carry out implantation process again.
The result of this process is that total natality that survives is 23%.Because the ES clone of using is planted from painted Holland's band, GM3 ES injection cell is come in will produce tangible crust color in mosaic in the blastaea of non-staining New Zealand white race.Judge according to the crust color, obtain having three chimaeric animals of (black-tape to Holland's band kind be typical) of one or more black-tape.The ratio of mosaic changes between more than 50% 10%, according to the contribution of Holland's band kind of painted crust.One is that male (Fig. 2 a), one is female (Fig. 2 b), and another may be a telianthus in these mosaics.These results have constituted first evidence of following principle, promptly by keeping in cultivation and the ES clone of (15 to 20 times) of going down to posterity is in a large number carried out the blastaea injection and can be produced chimeric rabbits.Recall, mosaic be it seems, further describes as following, owing to the alkaline phosphatase enzyme positive ES cell of three dimensional growth.
Table 2:ES injection cell advances chimeric generation behind the segmentation cavity of New Zealand's white race blastaea
The injection of # blastaea The ES cell that # goes down to posterity # natality (%) # mosaic (%)
??????76 ??????124 ??????87 ????20-30 ????50-100 ????100-300 22????30(41) 15????31(25) 18????6(7) ????1(1) ????1(1) ????1(1)
Sum 287 ????67(23) ????3(1)
Percentage in the bracket is a number of injecting blastaea relatively.
Really, after the ES injection cell advances segmentation cavity, the frequency that mosaic forms be low-have only 4% survive the birth animal.Perhaps, this is because following Several Factors, comprise 1) owing in the ripe blastaea that the ES cell will import significant redundant space is arranged, cause the ES cell often not to be integrated among the developmental embryo, 2) Holland band kind of ES clone and New Zealand's white race blastaea inherent uncompatibility, or 3) the losing of clone pluripotency.Attempt to improve with ES cell transfer inner cell mass in grow by the ES cell directly being imported developmental inner cell mass, be integrated into embryo's subsequently precondition, frequency, do not cause higher frequency or chimeric formation.Use Dutch white metal kind blastaea (originating from Holland's band by natural point mutation plants), to remove the species obstacle that involves like this, also do not produce mosaic as the acceptor of ES injection cell.
Further test discloses as Graves and the described ES cell that keeps in cultivation of Moreadith (1993) lack mosaic formation and as the low frequency of the ES cell that begins to cultivate from the GM3 cell that goes down to posterity in early days as described in the embodiment 1, most likely because the former lacks remaining pluripotency ES cell, the low percentage ratio of latter remnants' pluripotency ES cell.This point can disclose by alkaline phosphatase staining, and alkaline phosphatase exists in undifferentiated cell, in case break up then rapidly disappear (Benham etc., 1981).The existence of alkaline phosphatase positive cell in initiating cell system is less than 1% (Fig. 1) after 10 generations, although can be kept under the improvement culture condition that this frequency is described herein.Squamous epithelium like cell type (Fig. 1) great majority that show the ES cell (Graves and Moreadith, 1993) of thinking that originally representative is confirmed are alkaline phosphatase feminine genders, can not be integrated into the inner cell mass that is subjected to stomatoblastula, can not produce chimeric offspring.So press the new ES clone of embodiment 3 described derivation.The derivation of embodiment 3 improved ES cells
As described below, ES is cell-derived to improve one's methods developedly, and it and improved cell culture condition allow to be created in 5 rabbit ES clones that surpass 80% undifferentiated alkaline phosphatase positive cell through comprising after 8 to 10 generations together.
Holland band kind of the doe of hypervelocity ovulation and Holland's band kind of male rabbit mating.Go out blastaea at post-coitum 5.5 days (rather than post-coitum 4 or 5 days) from horn of uterus, wash with the phosphate buffered saline buffer of the Dulbecco that adds 3% bovine serum albumin (Cohn component V) and 5% (v/v) microbiotic/antimitotic solution.Blastaea contains 5%CO in 39 ℃ in rabbit ES substratum (as mentioned above) 2Incubator in preserve until further operation.With acidifying phosphate buffered saline buffer (pH2.5) be present in Saliva Orthana adventitia and the zona pellucida that 0.5% PRONASE A in the phosphate buffered saline buffer is removed blastaea.With two pins with inner cell mass from around trophectoderm cell artificial preparation come out, put into one by one 96 hole culture dish (with to be equivalent to each 10cm culture dish 3 to 4 * 10 612.5 days of individual cell density, the mouse embryo fibroblasts in 1 generation are the shop dish together).
The inner cell mass that shifts out is cultivated again with aforesaid improved rabbit ES cell culture medium (wherein the mouse leukemia supressor is substituted by people or Rab-LIF) every day.After 2 days, make the trophectoderm wart leave following feeder layer gently by using the glass pipette that curves the oblique angle, and, at an easy rate the inner cell mass wart is separated from remaining trophectoderm cell by its is sucked in glass pipette.
The clone who only with the three dimensional growth is the ES sample of feature is gone down to posterity in new culture dish subsequently, after 4 to 5 days, digest 96 holes with aforesaid tryptic digestion substratum selectivity, allow the ES cell like this, rather than trophectoderm cell or mouse embryo fibroblasts selectivity go down to posterity.Improve the typical three dimensional growth of rabbit ES cell and do not noticed also in the past that it obviously was a new feature of this class clone.Have only and use improved culture condition, and use 5.5 days embryo, three-dimensional undifferentiated ES cell clone could cultivated the high frequency maintenance.In order to keep the ES cell under very high density, another prevents to break up the precondition of losing with pluripotency, and the ES cell little by little is passaged in the big slightly culture dish with 4 to 5 days interval.Feeder cell keep each 10cm culture dish 3 to 4 * 10 mutually on culture dish subsequently 6The density of individual cell.
The rabbit ES clone (Fig. 3) that obtains by this process more is similar to pluripotency mouse ES cells system than any rabbit ES varient of reporting in the past.Principal character is clone's three dimensional growth, high refractive index and little nuclear-cytoplasmic ratio.Most important characteristic is after going down to posterity for 10 times, 80 to 90% of ES cell still keeps not differentiation, and this point proves by the negative staining (breaking up known mark) of alkaline phosphatase positive staining (Fig. 3 b) and people's cytokeratin 18 and mouse vimentin, (Viebahn etc., 1988; Piedrahita etc., 1990).These characteristics have formed the main difference (Fig. 1) with the ES clone of former affirmation (judging that by the alkaline phosphatase positive staining it is undifferentiated being less than 1% cell).In the rabbit ES of continuous passage cell, the phenomenal growth of undifferentiated cell ratio (from 1% to 80-90%) should significantly increase by the ES injection cell and advance blastaea, produces the efficient of chimeric rabbits, and allows to have the generation of the chimeric rabbits of target hereditary change.Reference
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-Sukoyan MA, Golubitsa AN, Zhelezova AI, Shilov AG, Vatolin SY, Maximovsky LP, Andreeva LE, Mc whir J, Pack SD, Bayborodin SI, Kerlkis AY, Kizilova HI, Serov OL (1992): separation and cultivation are from the stem cell line of the blastaea derivation of U.S. mink.Mol?Reprod?Dev33:418-431
-Talbot NC, Caird ER jr, Vernon GP, Powell AM, Nel ND (1993): the pig ectoderm cell of cultivating pig blastocyst.In?Vitro?Cell?Dev?Biol29A:546-554
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-Viebahn C, Lane BE and Ramackers FCS (1988) cell in the body early embryo of rabbit embryonic takes place has the expression of albumen and vimentin.Cell tissue research 253:553-562.
-Wilson JM (1996): the animal model of human disease that is used for gene therapy.J.Clin?Invest?97:1138-1141
-Yang X, Foote RH. (1998): from morula, produce chimeric rabbits by simple method.Gamete Res 21:345-351 diagram Fig. 1:
Rabbit ES cell (GM3 clone) by Graves and Moreadith derivation
A) phase-contrast microscopy of the ES cell of Que Rening has disclosed squamous epithelium sample phenotype
B) the ES cell alkaline phosphatase staining of Que Rening has shown the percentage of the alkaline phosphatase positive cell (redness) of low-down (<1%).Fig. 2:
A) have the male mosaic of a black-tape.
B) have the female sex mosaic of several black-tapes.
Band is the characteristic feature that Holland's band is planted, and GM3ES clone is by Holland's band kind of derivation.Fig. 3:
A) use the micrography that differs of the new clone of above-mentioned improved cell cultures and ES derivatization conditions deutero-.
B) alkaline phosphatase staining of above-mentioned improved cell cultures of use and ES derivatization conditions deutero-rabbit ES clone.New ES clone has three dimensional growth, the feature of high refractive index and 80 to 90% alkaline phosphatase positive stainings.

Claims (16)

1. rabbit embryonic is done (ES) clone, comprises at least 70%, 80 to 90% undifferentiated cells preferably, and the inner cell mass by separating 5.5 days blastaea of post-coitum and cultivating on the feeder cell in rabbit ES substratum obtains,
2. do (ES) clone according to the rabbit embryonic described in the claim 1, wherein rabbit ES substratum comprises the Eagle substratum of the modification of high glucose Dulbecco, the 4mM L-glutaminate, 0.1mM 2 mercapto ethanol, 148 units/ml penicillin G sodium salt, 148 milligrams/ml Vetstrep, 4 milligrams/ml Sigma I8405,10 3Unit/ml mouse leukemia supressor, 20% foetal calf serum, 1.5%MEM non-essential amino acid solution.
3. do (ES) clone according to the rabbit embryonic described in claim 1 or 2, wherein feeder cell are with in each 10cm petri plate 3 to 4 * 10 612.5 days mice embryonic feeder cell of individual cell density.
4. do (ES) clone according to the rabbit embryonic described in the claim 1,2 or 3, wherein the ES cell is through the cultivation of repeatedly going down to posterity, and before going down to posterity at every turn with selectivity tryptic digestion substratum tryptic digestion ES cell, this selectivity tryptic digestion substratum comprises 0.1% collagenase, 1% chicken serum and be present in 0.03% trypsinase-EDTA in the phosphate buffered saline buffer.
5. do (ES) clone according to any one described rabbit embryonic among the claim 1-4, it is characterized in that, after going down to posterity above 10 times, three-dimensional clone forms alkaline phosphatase positive staining and cytokeratin 18 and vimentin negative staining.
6. do (ES) clone according to any one the described rabbit embryonic among the claim 1-5, can in the generation of chimeric rabbits, use.
7. do (ES) clone according to any one described rabbit embryonic among the claim 1-5, be used for gene alteration by homology or non-homogeneous reorganization.
8. do (ES) clone according to any one described rabbit embryonic among the claim 1-5, be used for by kind being to transmit the rabbit that produces gene alteration.
9. do (ES) clone according to the rabbit embryonic described in the claim 1-5, the research that is used for (newly) gene with separate.
10. according to of the aborning application of the described ES clone of claim 1-5 at chimeric rabbits.
11. according to the application of the ES clone described in the claim 10, be used for blastaea be injected into moved by stomatoblastula or embryo cohesion or consideration convey after, produce chimeric rabbits.
12., be to be used on the same group or the gene alteration of non-homogeneous reorganization by homology according to the application of the described ES clone of claim 1-5.
13., be to be used for by kind being to transmit the rabbit that produces gene alteration according to the application of the described ES clone of claim 1-5.
14. according to the application or the differentiation of the described ES clone of claim 1-5, the research that is used for (newly) gene with separate.
15. rabbit ES substratum comprises the Eagle substratum of the modification of high glucose Dulbecco, 4mM L-glutaminate, 0.1mM 2 mercapto ethanol, 148 units/ml penicillin G sodium salt, 148 milligrams/ml Vetstrep, 4 milligrams/ml Sigma I8405,10 3Unit/ml mouse leukemia supressor, 20% foetal calf serum, 1.5%MEM non-essential amino acid solution.
16. selectivity tryptic digestion substratum comprises 1% collagenase, 1% chicken serum and be present in 0.03% trypsinase-EDTA in the phosphate buffered saline buffer.
CN97194183A 1996-04-29 1997-04-29 Pluripotent rabbit embryonic stem (ES) cell lines and their use in generation of chimeric rabbits Pending CN1217746A (en)

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