Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.Download video file.^{(61M, mov)}     

HSV-tk基因逆转录病毒重组体的构建与DNA序列分析

目的　构建含有单纯疱疹病毒Ⅰ型胸苷激酶 (HSV1 tk)基因的逆转录病毒重组载体pLXSN TK。方法设计一对寡核苷酸引物 ,用PCR方法从质粒pHSV10 6中特异扩增HSV tk基因片段 ( 1168bp) ,分别用BamHI和Eco RI酶切后 ,定向连接到质粒pLXSN中 ,转化宿主菌TG1,分别用上述内切酶 ,PCR和DNA测序鉴定重组质粒。结果　酶切鉴定所切下的片段和PCR扩增的片段大小均与预计相符 ,测序结果与文献报道序列及预计结果一致 ,证实符合表达框架。结论　成功构建了HSV tk嵌合重组质粒pLXSN TK。     

A Late Role for bmp2b in the Morphogenesis of Semicircular Canal Ducts in the Zebrafish Inner Ear

Background

The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

Methodology/Principal Findings

We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b^−/−) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.

Conclusions/Significance

Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.     

Effects of Mutations at the W Locus (c-kit) on Inner Ear Pigmentation and Function in the Mouse

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survivial of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (W^v/W^v) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (W^x, W^sh, and W⁴¹) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of W^sh/W^x, 25% of W^sh/W^sh, 60% of W⁴¹/W^x, and 69.2% of W⁴¹/W⁴¹ ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W⁴¹/W^x, W^sh/W^sh, and W⁴¹/W⁴¹ mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.     

Digital Reconstruction of the Otic Region and Inner Ear of the Non-Mammalian Cynodont Brasilitherium riograndensis (Late Triassic, Brazil) and Its Relevance to the Evolution of the Mammalian Ear

The external anatomy of the petrosal, the bony labyrinth of the inner ear, and the stapes of Brasilitherium riograndensis (specimen UFRGS-PV-1043-T) were investigated by digital 3D reconstructions based on μCT scan images. Brasilitherium is the most basal taxon bearing a distinct promontorium, although less inflated than that of Morganucodon and still lacking a flat medial facet. A bony wall formed by the petrosal separates the cochlear canal and the vestibule from the brain cavity, with an internal acoustic meatus bearing distinct foramina for the facial nerve (VII) and vestibulocochlear nerve (VIII). The semicircular canals are irregular in shape, the anterior canal being the largest and the lateral one the smallest. Brasilitherium has an elongated but straight cochlear canal. The stapes resembles the morphology of derived non-mammaliaform cynodonts, such as Probainognathus and Pachygenelus, and differs from Thrinaxodon. By the allometric relationship of the cochlear canal and the estimated body mass, Brasilitherium can be grouped with Yunnanodon and Morganucodon in a regression line, which is below the line of mammals and above the line of non-avian reptiles. Brasilitherium fits in a sequence of gradual elongation of the cochlear canal associated with the enhancement in the capacity to hear higher frequencies. Among the constraints that might have triggered these transformations in small, insectivorous, and possibly nocturnal Mesozoic cynodont taxa is the improvement of detecting acoustically active insects.     

Pargyline and р- Chlorophenylalanine Decrease Expression of Ptpn5 Encoding Striatal-Enriched Protein Tyrosine Phosphatase (STEP) in the Mouse Striatum

Striatal-enriched protein tyrosine phosphatase (STEP), which was initially identified in the striatum, is encoded by the Ptpn5 gene and is expressed in neurons of various structures of the brain. STEP is involved in regulating neuroplasticity, and its expression abnormalities are associated with human neurodegenerative disorders. The STEP inhibitor 8-trifluoromethyl-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153) has been shown to affect the serotoninergic system of the brain. However, the influence of the serotoninergic system on the STEP regulation has not been studied yet. The aim of the study was to investigate how pharmacologically induced changes in the brain serotonin (5-HT) level affect Ptpn5 expression and STEP activity in adult male C57BL/6J mice. To modulate the 5-HT level in the brain, the 5-HT synthesis inhibitor p-chlorophenylalanine or 5-HT degradation inhibitor pargyline was administered intraperitoneally for three successive days. Changes in 5-HT concentration in the brain were assayed using high-performance liquid chromatography. The STEP activity was determined spectrophotometrically in the supernatant by the rate of p-nitrophenyl phosphate dephosphorylation in the absence and presence of the selective STEP inhibitor TC-2153. The Ptpn5 mRNA level was determined using quantitative RT-PCR. The Ptpn5 expression level in the striatum was three times higher than in the cortex and hippocampus. Both increases and decreases in brain 5-HT were for the first time associated with a decrease in Ptpn5 mRNA in the striatum. STEP activity in the striatum and cortex was significantly higher than in the hippocampus. However, p-chlorophenylalanine and pargyline did not affect the STEP activity in the brain structures tested. Thus, a new method was proposed to study the STEP activity in the brain and p-chlorophenylalanine and pargyline were shown to decrease Ptpn5 expression in the striatum in mice.     

A Further Investigation of the Effects of Extremely Low Frequency Magnetic Fields on Alkaline Phosphatase and Acetylcholinesterase

Using a custom build spectrophotometer equipped with Helmholtz coils and designed to study the effects of magnetic fields on enzyme reactions in real-time we have investigated the influence of fields, from 100 μT to 10 mT and at a variety of field frequencies, on the membrane bound enzymes alkaline phosphatase and acetylcholinesterase. We have also employed other methods to apply a magnetic field, e.g. Biostim. In contrast to earlier reports we have been unable to detect any field effects on these enzymes under any field/frequency regime. We discuss possible reasons for the discrepancy between this and earlier work and note the particularly complex influence of small temperature changes that may confound analysis.     

Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo

Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.     

小鼠Myf5真核表达载体的构建及其在细胞中的定位

目的：获得小鼠生肌调节因子Myf5基因并构建pEYFP-C1真核表达载体,观察Myf5在小鼠C3H10T1/2细胞中的定位。方法：利用PCR获得Myf5基因克隆到pEYFP-C1载体中,利用脂质体将构建的表达载体转染C3H10T1/2细胞,荧光观察融合蛋白的表达。结果：从小鼠cDNA文库中得到760bp的myf5的CDS序列后,重组到pEYFP-C1载体中并转染C3H10T1/2细胞,荧光显示Myf5蛋白定位在细胞核中。结论：Myf5载体成功构建并在小鼠C3H10T1/2细胞表达,证明了Myf5蛋白定位于细胞核,为进一步研究Myf5与其他蛋白的相互作用奠定了基础。     

Protective Efficacy and Immunogenicity of an Adenoviral Vector Vaccine Encoding the Codon-Optimized F Protein of Respiratory Syncytial Virus

Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.Human respiratory syncytial virus (RSV) is a highly infectious member of the paramyxovirus family causing upper and lower respiratory tract infections in humans. Serious acute RSV infections, including fatal cases of bronchiolitis and pneumonia, occur particularly in premature infants, immunocompromised adults, and patients with pre-existing chronic lung diseases or underlying heart defects (11, 12, 14, 39, 46, 56). In young children, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of hospitalizations due to lower respiratory tract infections (21). In population-based surveillance studies for hospitalization in Europe, RSV was identified in 42 to 45% of enrolled children younger than 2 years with lower respiratory tract infections, and the rate of hospitalization due to RSV-induced respiratory illnesses was estimated at 3 to 6% among industrialized nations (45). Children with severe RSV infections suffer from oxygen deficiency with cyanosis and require intensive medical care. Furthermore, RSV infection in childhood is suspected to be a risk factor for development of asthma (36, 41, 43, 59). The urgent need for an RSV vaccine is further demonstrated by a study showing that levels of disease burden, mortality, and morbidity caused by RSV infections in the elderly are comparable to those induced by nonpandemic influenza A infections (11). However, the immunization of children with a formalin-inactivated (FI) RSV vaccine in the 1960s resulted in a more severe clinical illness, with two fatal cases, than in nonvaccinated infants following RSV infection, pointing out the difficulties in developing a safe and efficacious RSV vaccine (7, 29). It was shown previously that the enhanced disease severity and the development of pulmonary eosinophilia are mainly attributable to an excessive Th2-polarized immune response (15, 35, 57). Furthermore, the lack of high-affinity antibodies after poor Toll-like receptor stimulation has been suggested to be a key factor of the enhanced disease induced by FI RSV vaccination and subsequent RSV infection shown recently (8). However, the enhanced disease induced by FI RSV could partially be reversed by the chemical reduction of the carbonyl groups produced by prior treatment with aldehyde (34).Passive transfer of a neutralizing monoclonal antibody directed against RSV F (palivizumab) results in significant reduction of hospitalization rate due to RSV infection in children and preterm infants (16, 25), making RSV F a promising vaccine candidate for active immunization. Besides being a target for neutralizing antibodies, RSV F additionally contains cytotoxic-T-cell epitopes (1, 37). Moreover, RSV F based DNA vaccines induced encouraging immune responses of a balanced Th1/Th2 type in mice, as pulmonary eosinophilia and disease-enhancing effects were not observed after viral challenge (4, 5, 19, 31, 52). Additionally, RSV F is highly conserved between the two antigenic subgroups of RSV, which allows generation of cross-reactive antibodies after immunization (26).We recently showed that vaccination with codon-optimized RSV F expression plasmids induced improved humoral immune responses in mice compared to vaccination with wild-type cDNA expression plasmids (52). Consequently, viral load was reduced 13-fold in mice immunized with full-length RSV F and 170-fold in mice immunized with the soluble form of RSV F following RSV challenge in comparison to nonimmunized mice. Based on these results, we inserted the codon-optimized open reading frame (ORF) of both full-length RSV F and its soluble form into a replication-deficient adenoviral serotype 5 vector (AdV), generating AdV-F and AdV-Fsol, respectively, to further enhance the immunogenicity and efficiency of the delivered RSV F transgenes. AdVs were chosen because these viral vectors have been extensively studied and have proven their potential as vaccine vectors in multiple successful preclinical studies (reviewed in references 47, 24, and 51). AdVs are also potent inducers of both humoral and cellular immune responses in animal models and in humans (48, 49, 55). Furthermore, convenience of vector design, ease of handling and a robust antigen expression make AdVs a promising vaccine delivery platform. Another main advantage is their natural tropism for mucosal surfaces, which makes adenoviral vaccines convenient for the purpose of immunization against respiratory pathogens that preferentially initiate infection at the mucosal site (40).However, AdV vaccines expressing the wild-type RSV F protein were tested in several animal models without achieving convincing protection against RSV challenge (13, 22, 23). This might be due to poor RSV F expression levels caused by premature polyadenylation, which could be overcome by codon optimization (53). Hence, here we used the codon-optimized RSV F based AdVs AdV-F and AdV-Fsol and evaluated their potential as RSV vaccines, showing greatly improved vaccine efficacy.     

Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.     

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1^−/−;Lrig2^−/− double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1^−/−;Lrig2^−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member.     

A Mechanism by Which the Hair Cells of the Inner Ear Transduce Mechanical Energy into a Modulated Train of Action Potentials

Physical models of the hair cells of the inner ear were built and analyzed. These models suggest that a straightforward physical process is capable of modulating the electrical resistance of the hair cell. Strong evidence in the literature indicates that such a change in resistance would modulate an otherwise steady electrical current which flows across the hair cell. This would cause the resting potential of the hair cell to change in a systematic fashion, eventually giving rise to the modulated train of action potentials in the neurons leading from the hair cell to the central nervous system.     

跨膜蛋白基因克隆载体pMBL的构建及验证

自行设计一对引物,从质粒pUC18上扩增一段无启动子和信号肽的β-内酰胺酶基因(△P△SP Amp),以作为最终构建的载体克隆到带跨膜信号基因片段的报告基因。所设计的上游引物中依次带有分别处于3个不同阅读框架、且形成匹配粘性末端的3个酶切位点BglⅡ、BclI、BamHI,以便最终构建的载体捕获基因时的对位融合和表达。pET-28经BglI、Bst1107I双酶切,去除约2．5kh的lac I等基因的冗余片段,保留Kan抗性基因、复制原点、多克隆位点等结构,并消除BglⅡ位点,获得作为最终构建载体的抗性基因和基本骨架的过渡质粒pKan。△p△SP Amp基因经pGEM-T-EASY载体,克隆到pKan的EcoRI和XbaI间,得到在Kan平板中生长而在Amp和Kan双抗平板中不能生长的转化子pMBL-E质粒;经部分酶切补平自连,筛选得到消除HindⅢ位点端EcoRI位点的质粒,即得到用于跨膜蛋白信号基因片段捕获克隆的目的载体pMBL,大小为3．46kb。经酶切鉴定和测序,证明构建的载体与预期设计的一致。应用四环素抗性基因(Tet)片段对构建的跨膜蛋白基因克隆载体pMBL．的有效性进行了验证,在克隆人EcoRI和BglⅡ双酶切(0位)的载体中,Kan和Amp双抗平板筛选到阳性克隆子,经酶切和测序均显示Tet基因已对位插人,并启动了β-内酰胺酶的表达和跨膜分泌。由此证明：构建的跨膜蛋白克隆载体pMBL能有效捕获含启动子和信号肽序列的跨膜蛋白基因。     

妊娠晚期腹腔注射酸性磷酸缓冲液对两个近交系小鼠繁育生理影响的对比分析

目的对比分析妊娠晚期腹腔注射缓冲液对近交系SPF级C57BL/6J（B6）和BALB/c（B/c）小鼠繁育生理的影响。方法 B6和B/c小鼠随机、全同胞兄妹2∶1/1∶1（♀/♂）过夜同居交配,观察交配方式、阴栓与受孕率的关系;受孕鼠在妊娠的第17.5天（妊娠0d=交配后当天）接受腹腔注射酸性磷酸缓冲液,观察注射对母鼠及胚胎的作用及子鼠从离乳到8周的早期生长发育情况。结果 B6较B/c雌鼠的受孕率高（29.4%vs.21.1%[2∶1],33.2%vs.29.7%[1∶1]）;交配后10 d～14 d,根据雌鼠增大的腹部、结合体重来判断受孕较观察阴栓更为准确;比较而言,妊娠晚期腹腔注射对B6母鼠及胚胎的影响较大,表现在离乳子鼠数量减少（4.7±3.1 vs.6.1±2.1,P=0.231）,离乳时两性别的子鼠体重（g,雌性：11.7±1.1 vs.12.7±1.5;雄性：12.8±1.3 vs.13.6±1.5）显著降低（P均〈0.05）及两品系子鼠早期生长发育的方式显著不同（P=0.000）。结论近交系小鼠繁殖生理存在品系差异;两品系受孕鼠对妊娠晚期腹腔注射的耐受不同,并可能影响实验动物产后的哺乳过程及子鼠的早期生长发育。