Linearization and purification of BAC DNA for the development of transgenic mice

Bacterial artificial chromosome (BAC) vectors are increasingly used for generation of transgenic mice due to the relatively large size and the stability of their inserts compared to YACs. We have compared methods for purification and linearization of BACs, and describe an optimised protocol for preparation of high quality linear BAC DNA based on terminase digestion, electroelution of linearized DNA together with simple preliminary multiplex PCR screening to detect transgenic mice. Linearized BAC DNA purified this way was successfully used for the development of transgenic mice containing 2–4 copies of the transgene.     

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes.

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue-specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto-oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non-acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto-oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting E mu-pim-1 and E mu-myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种快速鉴定转基因植物纯合体的新方法

植物转化中鉴定转基因植物的整合性是一个很重要的步骤,常规方法是对独立分离的转基因T1代植株产生的T2代进行转基因分离比率研究,以检测T1代的转基因整合状态,不仅费时费力,而且浪费了T1代资源。本介绍一种应用双重定量实时PCR技术鉴定转基因植物纯合子的新方法：以T1代植物DNA为模板,根据转基因后代的Ct表型值鉴定其转基因整合状态,Ct值接近2的为转基因纯合型,Ct值接近1的为转基因杂合型。用这种方法,可以同时对数十个T1代转基因幼苗的整合状态进行快速鉴定,准确率为100％。     

小鼠精子形成各阶段转基因效率的研究

在过去的近30年中,转基因技术在哺乳动物基因表达方面研究的应用已经成为实验生物学及应用生物学领域最为显著的进展之一．传统的制作转基因动物方法有显微注射法、逆转录病毒感染法和胚胎干细胞法等,但每种方法都有其缺陷,限制了其在今后转基因动物研究中的广泛应用．对小鼠体内生殖细胞进行外源基因转染,研究精子形成过程中制作转基因小鼠的效率．首先运用睾丸注射法将被脂质体包裹的绿色荧光蛋白表达载体(pIRES2-EGFP)注射到公鼠睾丸及附睾内,然后根据精子形成的不同阶段,分别于注射后7、16、30和42天与发情母鼠合笼,利用PCR和DNA印迹方法对新生小鼠进行基因组DNA检测．在各阶段所得新生小鼠中PCR阳性率分别为6.82%、0、56.86%和42.86%,DNA印迹检测阳性率分别为6.82%、0、47.06%、34.69%．经活体荧光成像系统及荧光显微镜分析,转基因小鼠呈现绿色荧光表达．通过比较精子生成各阶段转基因效率高低,为以后通过用睾丸内注射法转染雄性生殖细胞高效制作转基因动物提供了理论依据．     

An efficient method of generating transgenic mice by pronuclear microinjection

Genetic transformation of mice using pronuclear microinjection was demonstrated by a number of groups in rapid succession in the early 1980’s. Since that time, studies using transgenic animals have produced major advances in biomedical sciences and molecular genetics. More important, it is possible to study the molecular basis for tissue and stage-specific expression of genes. We recently used this method to generate transgenic mice. DNA fragment (transgene) was injected into the pronucleus of one-cell embryos. We describe this simplified protocol, which is reliable. With the use of buffered medium M2 for the whole process, it is not mandatory to have a CO₂ incubator.     

转基因小鼠乳腺表达G-CSF的研究

用PCR法从正常中国人脐带血提取总DNA作为模板,扩增出1.5 kb的人G-CSF基因组基因。序列分析证实其正确性。将其插入小鼠乳清酸蛋白(WAP)基因的起始密码子ATG前的KpnⅠ位点,使其受控于2.6kb的WAP调控序列,构建成乳腺表达载体pWGG。回收经EcoRⅠ酶切后的8.7kb片段用于显微注射。共注射1200枚受精卵,移植34受体母鼠,产仔鼠85只。经PCR检测和DNA印迹分析,证实获得两只整合有人G-CSF基因的雄性鼠,整合率为2.37％。建立的转基因鼠系表明,采用ELASA方法对F1代雌鼠乳汁检测,成功地表达出人G-CSF。表达量为120～250ng/ml。这一结果表明转基因的表达具有乳腺特异性。这为在大动物中实施转基因提供了依据。     

利用转基因小鼠及转染色体小鼠产生人抗体的研究进展

自单克隆抗体（mAb)技术问世以来,解决了生命科学的许多重要问题,但其鼠源生导致的HAMA反应在大大限制了它在人体治疗中的应用,因此,制备人抗体成了亟待解决的难题,转入Ig基因组小鼠与转梁色体小鼠 的构建成功为解决这一难题提供了可行途径,本文就这两条小鼠的构建及其在制备人抗体中的应用进展作一综述。     

Expression of Nkx2-5-GFP bacterial artificial chromosome transgenic mice closely resembles endogenous Nkx2-5 gene activity

Mouse Nkx2-5 gene is essential for early heart development and it is regulated by a complex array of regulatory modules. In order to establish an efficient in vivo system for mapping the Nkx2-5 genomic locus for regulatory regions, we developed improved homologous recombination technology for use in Escherichia coli and then knocked an IRES-hrGFP reporter gene into Nkx2-5 gene in a 120 kb Nkx2-5 bacterial artificial chromosome (BAC) clone. We employed the recombination genes redalpha and redbeta under the pBAD promoter, which was specifically induced by the addition of L-arabinose. Recombination was selected for by our universal targeting cassette which conferred kanamycin resistance in bacterial cells and neomycin resistance in mammalian cells. Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. BAC transgenics using the GFP reporter offers an efficient model system to study gene expression and regulation.     

TrackingH-2 alleles in transgenic mice by RFLP and heteroduplex analysis

Transgenic mice provide valuable tools for biological research including many areas of immunology. In studies involving the major histocompatibility complex (MHC), it is often necessary to place the desired transgene in a specificH-2 (the murine MHC) environment. In this regard, the strains commonly used for the production of transgenic mice also carry well characterizedH-2 alleles and provide an appropriate genetic background for MHC related experiments. In this study, a highly polymorphic microsatellite of tetranucleotide repeats from the second intron of the class IIEb gene within theH-2 complex was used in order to identify the corresponding alleles. The relevantH-2 allele(s) along with the transgene were then tracked throughout the production of a chicken ovalbumin-specific transgenic strain. The technique involved PCR-amplification of a DNA sequence encompassing theH-2 specific microsatellite followed by RFLP and heteroduplex analyses. This approach is likely to find wide application in the background checking of trangenic mice, especially in immunological research requiring a definedH-2 background.     

FISH mapping and sequence analysis of 87 porcine BAC clones

Ninety-one bacterial artificial chromosomes (BAC) clones, selected effectively at random from our library, were used as probes for fluorescence in situ hybridization. Of these, 87 clones gave a specific signal in one, two or three different pair(s) of swine metaphase chromosomes. The ends of 35 BAC clones were sequenced in order to obtain information for comparative mapping. Fifteen of them gave useful comparative mapping information.     

绿色荧光蛋白转基因小鼠模型的建立及胚胎冷冻保种

目的建立绿色荧光蛋白转基因小鼠模型,并采取胚胎冷冻的方法进行保种。方法通过原核显微注射法,把线性化、纯化后的外源基因pEGFP注射入BDF1小鼠受精卵中,胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。经鉴定对有表达的转基因鼠进行胚胎冷冻保种。结果移植注射胚胎385枚给30只假孕小鼠共出生了306只后代鼠,经PCR和southern blot检测得到5只阳性小鼠。F2代转基因鼠胚胎冷冻240枚胚胎。结论通过显微注射法使外源基因pEGFP在小鼠基因组中得到整合,建立了转pEGFP的转基因小鼠模型。     

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y₁R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y₁R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09792-9.     

Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice

The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.     

Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice

The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation. The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized. These formulations were injected in mice transgenic (TG) for human genes for Ig heavy and light chains and their non-transgenic (NTG) counterparts. Anti-drug antibody (ADA) titers were determined by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels. A mild antibody response was obtained in a fairly small percentage of mice, when injected with shaken, pH-shifted and heated formulations. The metal-catalyzed oxidized IgG formulation was the most immunogenic one, in both ADA titers and number of responders. The overall titers of NTG responders were significantly higher than the ones produced by TG mice, whereas there was no significant difference between the overall number of TG and NTG responders. This study reinforces the important role of protein aggregates on immunogenicity of therapeutic proteins and provides new insight into the immunogenic potential of different types of IgG aggregates. The results indicate that the quality of the IgG aggregates has more impact on the development of an immune response than their quantity or size.     

含P301L突变的Tau转基因小鼠纯合子品系的建立及鉴定

目的:建立含P301L突变的tau转基因小鼠的纯合子品系。方法:雄原核显微注射法获得含P301L突变的tau转基因阳性首建鼠,通过SYBR Green实时荧光定量PCR法和传统育种方式结合鉴定纯合子和杂合子。结果:共选育出95只纯合子,鉴定出的纯合子具有优于杂合子模拟老年痴呆生物学特性改变的优势。结论:外源性基因tau能稳定遗传,采用的SYBR Green实时荧光定量PCR和传统育种方式结合筛选鉴定纯合子和杂合子快速、经济、可靠。     

Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue-specific gene expression.

Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.