源于129S1品系的小鼠胚胎干细胞系的建立及其生物学特性分析

从129S1小鼠早期胚胎的内细胞团分离、培养类胚胎样细胞,经反复传代,成功地建立了129S1小鼠胚胎干细胞系,命名为NM-2细胞系。形态学鉴定具有胚胎干细胞的典型形态特征,正常核型率为80％;呈碱性磷酸酶阳性、表达胚胎干细胞特异性转录因子OCT-4;体内分化后可形成源于三胚层的组织结构;经囊胚腔显微注射后所获得的子代个体中79％具有毛色嵌合表型;雄性嵌合个体中31％发生生殖腺嵌合;同时,通过育种观察到所有生殖腺嵌合体的子代小鼠表型正常。以上结果证实NM-2细胞系为一株具高生殖腺嵌合能力的小鼠胚胎干细胞系。     

Unequal Rates of Cell Proliferation in Tetraparental Mouse Chimaeras Derived by Fusion of Early Embryos

In spite of fusion at the early morula stage, tetraparental mouse chimaeras rarely reflect 50:50 mixtures of the two parental strain cell populations. Apart from this, changes are known to occur during life and on occasions this can result in the loss of one or other parental strain cell components.
In spite of roughly 'balanced' mixed coat colour composition and distribution of the gametes, cytogenetic analysis recently revealed an overwhelming preponderance of AKR mitoses in a group of tetraparental AKR↔CBA/H-T6 mouse chimaeras. Cytogenetic analysisis necessarily restricted to cells in division and therefore it was conceivable that this phenomenon may have only represented excessive AKR cell division balanced by an increased rate of AKR cell death. This view now seems unlikely since analysis of both serum allotypes and the red cell isoenzyme glucose phosphate isomerase (GPI) also showed a similar and overwhelming preponderance of AKR cell products. Factors responsible for excessive AKR cell proliferation remain to be determined. However, it should be noted that these were only effective after coat colour and differentiation of the gametes are established.     

利用胚胎干细胞分化的拟胚体研究小鼠早期胚胎发育过程

小鼠胚胎干细胞(ES细胞)具有分化的全能性已经得到广泛共识。ES细胞在体外分化所形成的拟胚体在结构上能够模仿早期胚胎发育过程,包括在内细胞团表面形成内胚层、柱状上皮细胞的分化,以及中央空腔的形成。本文介绍利用拟胚体研究小鼠早期胚胎发育过程中各个胚胎阶段的发育、细胞程序性死亡的发生及TGF-β信号在胚胎发育过程中的作用。     

3个不同品系小鼠ES细胞的绿色荧光蛋白标记

用电穿孔法将线性化的质粒pEGFP—N3分别导入来自129／ter、C57BL/6J和BALB／c3个品系的小鼠胚胎干细胞：MESPU—13、MESPU—35和MESPU—62中,经G418筛选、荧光显微镜镜检、阳性克隆扩增、流式细胞仪分选、再扩增以及核型分析等过程,分别得到核型大于85％的被EGFP稳定标记的细胞株5个(129／ter2个、C57BL/6J1个、BALB／c2个),分别命名为MESPU—13／G1和MESPU—13／G2、MESPU—35／G1、MESPU—62／G1和MESPU—62／G2。从不同品系中各选一个增殖生长快、形态典型的标记细胞株,进行碱性磷酸酶染色、oct4群基因产物的表达检测、类胚形成和体内分化鉴定,结果表明所得到的核型正常的、稳定标记的ES细胞系具有原ES细胞的典型特征。     

四倍体补偿技术的建立及其应用

常规基因剔除小鼠的获得主要是利用ES细胞的全能性先获得嵌合体小鼠,再利用:ES细胞的生殖系传递能力,通过嵌合体与野生型小鼠的交配获得杂合子小鼠.而四倍体补偿技术则可绕过嵌合体小鼠阶段,直接获得基因修饰杂合子小鼠.利用电融合技术和Piezoelectric microinjecfion显微注射技术建立了四倍体补偿技术,小鼠四倍体胚胎的获得率(电融合率)为(93.01±l.37)%,经体外培养囊胚形成率为(82.49±2.08)%.通过显微注射方法将2种129品系小鼠来源的ES细胞(CJ7和SCR012)注射到四倍体囊胚腔中,获得了完全ES细胞来源的小鼠,ES鼠的获得率分别为2.7%和8.3%.经微卫星DNA检测,成体小鼠的10个被检测组织均为129小鼠来源的.同时,也利用基因修饰的ES细胞进行了研究,获得了2种基因修饰的完全ES细胞来源的杂合子小鼠,部分小鼠具有繁殖能力,经繁育已获得了纯合子,其中凝血因子Ⅷ基因敲除小鼠获得了预期的血友病小鼠表型.上述结果说明四倍体补偿技术可应用于基因修饰小鼠的制备.     

Initiation of the Primary Immune Response to Sheep Red Blood Cells in the Dissociated Mouse Spleen Cell Culture

From a suspension of mouse spleen cells were separated two functionally different cell types on the basis of their ability or inability to adhere to plastic dishes during a short period of incubation. Morphological observations of cells of these two fractions were made with the aid of histochemical methods. The majority of cells in the adherent fraction possessed β-glucuronidase, which is one of the lysosomal enzymes rich in macrophages or phagocytic cells. In contrast, almost all cells in the non-adherent fraction were devoid of this enzyme activity and identified morphologically as small lymphocytes. The adherent and non-adherent cells were found to associate in cell clusters during the cultivation. In the cultures stimulated with sheep red blood cells, some of the non-adherent cells which were located peripherically in the cell clusters began to show alkaline phosphatase activity in their cytoplasm. This may perhaps indicate that antibody synthesis is going on in these alkaline phosphatase-positive cells, since in an in vivo study such cells were found to arise in the lymph nodes of immunized animals concomitantly with the appearance of specific serum antibody.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

C57BL/6 and AKR mice were treated with hamster erythrocytes (HRBC) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and the development of delayed hypersensitivity and antibody production were examined. 1) Delayed hypersensitivity against HRBC antigen, as determined by the peritoneal macrophage disappearance test, was detected in mice sensitized with HRBC in CFA but not in those sensitized with HRBC in IFA. 2) Antibody production against HRBC or hapten TNP after a booster injection of HRBC or trinitrophenylated HRBC (TNP-HRBC) in saline was enhanced by pretreatment with HRBC in CFA or IFA. 3) Delayed hypersensitivity was not detectable after a booster sensitization with HRBC in CFA in mice which had been pretreated with HRBC in IFA 2 weeks earlier. In the mice treated with both HRBC in IFA (day ?21) and in CFA (day ?7), however, an enhanced antibody production against HRBC or TNP was detected after an intravenous injection with HRBC or TNP-HRBC in saline (day 0). These results suggest that sensitized effector lymphocytes in delayed hypersensitivity and helper cells in antibody production may be derived from the same pool of unprimed T cells. The pool of unprimed T cells with a capacity to differentiate into either type of primed T cells may be exhausted after pretreatment with the antigen in IFA, and the primed helper T cells may not be able to differentiate into sensitized lymphocytes even after sensitization with the antigen in CFA, which favors development of delayed hypersensitivity in normal controls.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Antibody response against hamster red blood cells (H-RBC) was examined in inbred strains of C57BL/6, AKR, C3H/He, DDD and SL mice, and outbred CF1 mice. 1) There were strain differences in antibody response after a primary intravenous injection of H-RBC. DDD, SL and CF1 mice belonged to high-responder strains, while C57BL/6, AKR and C3H/He to low-responder strains. In the spleens of immunized CF1 and SL, 40 to 70 times as many plaque-forming cells (PFC) as those in C57BL/6 mice were detected. The magnitudes of the response were: CF1 ≒ SL>DDD>>C3H/He ? AKR>C57BL/6. 2) 2-mercaptoethanol resistant (MER) antibody was detected in neither low- nor high-responders after a primary intravenous antigen-injection. 3) After a secondary intravenous antigen-injection, MER antibody was detected in all the SL mice, but only in 30 to 50% of AKR and C57BL/6 mice. 4) A subcutaneous injection of H-RBC in Freund's complete adjuvant (FCA) did not elicit antibody production within 10 days. When mice pre-sensitized 7 days in advance w^Tith H-RBC in FCA were intravenously injected with H-RBC, enhanced antibody production of the primary type was observed in all the mouse strains. 5) In pre-sensitized mice, the extent of the enhancement of antibody production was the highest in low-responder C57BL/6 mice and the lowest in high-responder SL and CF1 strains. Thus, there was no strain difference in antibody titers or the numbers of PFC after the booster.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

In a previous paper we reported that an inbred strain of SL mice and an outbred strain of CF1 mice belonged to the high-responder strains in antibody production after primary immunization with hamster erythrocytes (H-RBC), while inbred strains of C57BL/6, AKR and C3H/He mice belonged to low-responder strains. In the present study we obtained the following results. 1) Pre-sensitization with hamster lymphoma enhanced antibody production after an intravenous injection of H-RBC. There was no strain difference in the pattern of antibody production against H-RBC among pre-sensitized mice. 2) The pattern of enhanced antibody production after an intravenous injection of H-RBC into pre-sensitized mice assumed the primary type in terms of time of appearance of hemolysin plaque-forming cells (PFC) in the spleens and the conversion from 2-mercaptoethanol sensitive to 2-mercaptoethanol resistant antibody production, when the intervals between both treatments were within 7 days. 3) Pre-sensitization with lymphoma induced not only an increase in numbers of PFC after an intravenous injection of H-RBC, but also an increase in the size of the hemolysin plaques. These results suggested that sensitization with hamster lymphoma stimulated some kinds of immuno-competent cells, which could contribute to antibody production against H-RBC after a booster injection of H-RBC.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Patterns of proliferation of antibody-forming cells after an intravenous immunization with hamster erythrocytes (HRBC) were compared in groups of mice possessing different activities of thymus-derived lymphocytes (T cells). 1) Marked differences in the numbers of hemolysin plaque-forming cells (PFC) after HRBC injection were found among the low- and high-responder normal mice and those pretreated with HRBC in complete Freund's adjuvant (CFA) or incomplete adjuvant (IFA), and they appeared to depend primarily upon the different rates of proliferation of antibody-forming cells rather than on the numbers of antigen-specific lymphocytes initiating the antibody response. 2) The numbers of hemolytic foci were slightly larger in mice with large numbers of PFC (normal SL mice, the pretreated SL and C57BL/6 mice) than in those with small numbers of PFC (normal C57BL/6 mice). The numbers of hemolytic foci increased at almost the same rate from day 2 to day 3 in both groups, while the numbers of PFC increased more efficiently in mice with large numbers of PFC than in those with small numbers of PFC from day 2 to day 3. Individual hemolytic foci appeared to contain larger numbers of PFC in mice with large total numbers of PFC than in those with small total numbers of PFC. 3) The numbers of rosette-forming cells (RFC) were increased by pretreatment with HRBC in CFA and by pretreatment with HRBC in IFA to almost the same extent. Rates of increases in PFC were, however, larger by pretreatment with HRBC in CFA than with HRBC in IFA. These results suggested that the activity of the T cell determined not only the rates of proliferation of antibody-forming cells but also the antibody-producing capacity of each cell.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Golden hamsters were used as hosts in this work, and mice of various strains as donors of antigens. 1) There were no strain differences in immunogenicity of erythrocytes from C57BL/6, AKR, SL and CF1 mice. 2) Primary intravenous immunization with mouse erythrocytes (MRBC) induced the production of hemolysin plaque-forming cells (PFC) in a large number, but elicited only in a negligible titer production of 2-mercaptoethanol-resistant antibody. 3) 2-Mercaptoethanol-resistant antibody was produced more efficiently in hamsters pre-sensitized with mouse lymph node (MLN) cells rather than those pre-immunized with MRBC after a booster with MRBC. 4) Numbers of PFC in pre-sensitized hamsters were three-times that of the non-sensitized hamsters after a booster with MRBC, when pre-sensitization was performed intradermally with a small number of MLN cells. 5) Average diameter of the hemolysin plaques in pre-sensitized hamsters was one and a half times larger than that in non-sensitized hamsters. Conclusions agree well with the results in our previous papers that the reversed combination of hosts and antigen donors employed support the concept that certain processes required for delayed hypersensitivity contributed to antibody production under a condition suitable for antibody response.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

The production of anti-hapten antibody after immunization with trinitrophenylated (TNP) hamster erythrocytes (HRBC) or sheep erythrocytes (SRBC) was determined in high- and low-responder mouse strains against HRBC antigen. 1) Anti-TNP antibody was detected in sera of high-responder DDD and CF1 mice after primary immunization with TNP-HRBC, but not in those of low-responder C57BL/6 mice. 2) Anti-TNP antibody was detectable in sera of all the strains after primary immunization with TNP-SRBC. 3) Production of anti-TNP antibody was elicited after a booster injection of TNP-HRBC in low-responder C57BL/6 mice pre-sensitized with HRBC in Freund's complete adjuvant. These results suggest that functions of thymus-derived cells specific for HRBC antigen are deficient in low-responder mice.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

1) A subcutaneous injection of hamster erythrocytes (HRBC) in Freund's complete adjuvant (FCA) or an intravenous injection of hamster lymph node (HLN) cells suppressed antibody production against HRBC in the low-responder C57BL/6 and AKR mice, when HRBC in saline were given on the same day; 2) The suppressing effect of such treatments was neither detectable in the high-responder SL mice, nor in the C57BL/6 mice, which had been pre-sensitized with HRBC in FCA or hamster lymphoma cells; 3) Positive reactions of the peritoneal macrophage disappearance test and the enhanced antibody production were detected seven days after treatment with HRBC in FCA and HRBC in saline, or HLN cells and HRBC in saline; 4) The suppressing effect of such simultaneous treatments on anti-HRBC antibody production was eliminated by a transfer of normal syngeneic thymus cells to AKR mice or a transfer of thymus cells from SL to C57BL/6 mice. Suppression of the antibody production in the low-responder mice by the described simultaneous treatments may be due to a competitive involvement of HRBC-specific thymus-derived cells (T cells) in the developmental stages of delayed hypersensitivity and antibody production. High-responder SL mice appear to have enough T cells for development of the delayed hypersensitivity and as helper cells in antibody production. These results appear to support the concept that T cells for delayed hypersensitivity and antibody production to HRBC antigen are derived from the same original pool.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Delayed hypersensitivity against hamster erythrocyte antigen was examined after sensitization with hamster erythrocytes (HRBC) in Freund's complete adjuvant (FCA). Extent of the delayed hypersensitivity was determined by the migration inhibition test, the peritoneal macrophage disappearance test and the skin test in the ear using solubilized HRBC as the test antigen. 1) Delayed hypersensitivity against HRBC developed earlier in high-responder SL mice than in low-responder C57BL/6 mice after sensitization. The period required for development of the delayed hypersensitivity in AKR mice was intermediate between periods in high-responder SL mice and low-responder C57BL/6 mice. 2) After sensitization with HRBC in FCA, a delayed hypersensitive state without detectable antibody production persisted until day 12 in high-responder SL mice and until day 16 or later in low-responder C57BL/6 mice. 3) Delayed hypersensitivity against HRBC antigen persisted even after the appearance of circulating antibody which occurred late after sensitization with HRBC in FCA or after intravenous injection of HRBC into sensitized mice.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

It was confirmed by passive transfer experiments that the function of thymus-derived cells specific for hamster erythrocytes (HRBC) was deficient in the low-responder mouse strains. 1) Antibody production against HRBC was enhanced by passive transfer of thymus cells from normal SL mice (high-responder) to normal C57BL/6 mice (low-responder). 2) The enhancing effect of passive transfer of thymus cells from SL mice was abrogated by pre-sensitization of the recipients (C57BL/6) with thymus cells from SL mice. 3) In C57BL/6 mice, antibody production against HRBC was enhanced by the transfer of lymph node or spleen cells from C57BL/6 mice which had been sensitized with HRBC in Freund's complete adjuvant or hamster lymphoma cells.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Enhancing and suppressing effects of microbial adjuvants were studied in female mice of the C3H/He, AKR and SL strains. Propionibacterium acnes, Bordetella pertussis, BCG and yeast cell wall (YCW) were chosen as adjuvants. As antigens, we chose hamster erythrocytes (HRBC) which proved to be a weak antigen for mice. Adjuvants were given on day —7, day 0 or day 3, and HRBC were injected on day 0. The results were as follows. 1) P. acnes facilitated IgM and IgG antibody production in AKR mice and suppressed IgM antibody production in SL mice, when given on day —7. When P. acnes was given on day 0, they suppressed IgM antibody production in all of the strains used. 2) When B. pertussis was given on day 0, it exhibited enhancing effects on IgG antibody production in all of the strains and a suppressing effect on IgM antibody production in SL mice. 3) BCG suppressed IgM antibody production in all strains when given on day 0. 4) YCW showed no influence on antibody production in any combination used in this work. 5) SL mice were very sensitive to suppressing effects by adjuvants. Strain differences in the expression of enhancing and suppressing effects by adjuvants appear to be under some control independent of antigen-specific immune response genes.     

潮霉素抗性3T3细胞的建立和鉴定

目的　建立具有潮霉素 (hygromycin)抗性的 3T3细胞系 ,用于转染目的基因 (pTRE Ins human)的ES阳性细胞克隆筛选的饲养层。方法　通过脂质体转染的方法 ,将含有潮霉素B磷酸转移酶基因的质粒pHyg导入 3T3细胞中 ,利用潮霉素的药物选择特性 ,对转染细胞进行压力筛选 ,并对其进行PCR鉴定。结果　经 5 0 0 μg ml的潮霉素压力筛选后 ,获得了抗性细胞克隆。抗性 3T3细胞的形态和生长速度与正常 3T3细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了潮霉素抗性的 3T3细胞 ,为进行目的基因 (pTRE Ins human)转染ES细胞的阳性细胞克隆筛选奠定了基础。     

用饲养层分离胚胎干细胞集落的实验初探

目的 用饲养层分离胚胎干细胞集落。方法 用胚龄为13~14 d的小鼠胚胎分离原代成纤维细胞,制成饲养层,用于囊胚的培养。结果 小鼠原代胚胎成纤维细胞（PMEF）贴壁能力较好,增殖快,易铺层。囊胚和内细胞团（ICM）在饲养层上贴壁生长良好,当培养4~5 d时,其增殖率为16/28（57%）。在ICM离散48 h后,各种胚胎干细胞（ES）集落开始出现。此种集落经碱性磷酸酶染色成阳性。结论 用饲养层分离胚胎干细胞获得初步成功。     

Molecular Characterization of Mutant Mouse Strains Generated from the EUCOMM/KOMP-CSD ES Cell Resource

The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3′ LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3′ LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.