Assessing somatic hypermutation in Ramos B cells after overexpression or knockdown of specific genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination. Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η). However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes. Ramos - a Burkitt lymphoma cell line that constitutively undergoes SHM - has been a popular cell-line model to study SHM. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).     

Differential protein expression in MCF7 breast cancer cells transfected with ErbB2, neomycin resistance and luciferase plus yellow fluorescent protein

Gene transfection is frequently used to explore the molecular and phenotypic consequences of introduced genes. Breast cancer cell lines transfected with genes for growth factor receptors, intracellular signaling molecules or genes that generate luminescent signals are widely used in basic science and preclinical studies. Typically, a target gene of interest is co-transfected with selectable markers that are generally assumed to be innocuous. Perturbations of the cellular genome by transfected sequences may induce subtle and/or unexpected modulations in protein expression, only some of which may be attributable to the target gene of interest. In this study, we show that neomycin resistant MCF7 cells (MCF7 Neo(r)) proliferate twice as rapidly in nude mice as do the untransfected parent cells, but show similar growth rates in vitro. MCF7 transfected with the ErbB2 gene shows minimal alteration in growth rate in vitro, and approximately a threefold increased growth rate in vivo. MCF7 cells that express luciferase and yellow fluorescent protein proliferate slowly in vitro and show essentially no growth in vivo suggesting that overexpression of these tracking proteins adversely affects cellular proliferative capacity. The molecular basis for alterations in proliferative capacity of the transfected sub-lines is poorly understood. We performed two-dimensional gel electrophoresis (2-DE) to compare relative protein expression among the cell lines. Relative to the parental MCF7, transfected cell lines displayed numerous differentially expressed proteins (69 to 149), relative to parental MCF7. Twenty-one of these differentially expressed proteins were identified by mass spectrometry, and included metabolic, structural, and signaling proteins. Possible roles of differentially expressed proteins in altering cellular proliferation are discussed.     

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

Conflict theory of genomic imprinting in mammals

In mammals, some embryonic genes are expressed differently depending on whether they are inherited from the sperm or egg, a phenomenon known as genomic imprinting. The information on the parental origin is transmitted by an epigenetic mark. Both the molecular mechanisms and evolutionary processes of genomic imprinting have been studied extensively. Here, I illustrate the simplest evolutionary dynamics of imprinting evolution based on the “conflict theory,” by considering the evolution of a gene encoding an embryonic growth factor controlling the maternal resource supply. It demonstrates that (a) the autosomal genes controlling placenta development to modify maternal resource acquisition may evolve a strong asymmetry of gene expression, provided the mother has some chance of accepting multiple males. (b) The genomic imprinting may not evolve if there is a small fraction of recessive deleterious mutations on the gene. (c) The growth-enhancing genes should evolve to paternally expressed, while the growth-suppressing genes should evolve to maternally expressed. (d) The X-linked genes also evolve genomic imprinting, but the main evolutionary force is the sex difference in the optimal embryonic size. I discuss other aberrations that can be explained by the modified versions of the basic model.     

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.     

CRISPR/Cas9‐mediated gene knockout in the ascidian Ciona intestinalis

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.     

Targeting of somatic hypermutation

Somatic hypermutation (SHM) introduces mutations in the variable region of immunoglobulin genes at a rate of approximately 10(-3) mutations per base pair per cell division, which is 10(6)-fold higher than the spontaneous mutation rate in somatic cells. To ensure genomic integrity, SHM needs to be targeted specifically to immunoglobulin genes. The rare mistargeting of SHM can result in mutations and translocations in oncogenes, and is thought to contribute to the development of B-cell malignancies. Despite years of intensive investigation, the mechanism of SHM targeting is still unclear. We review and attempt to reconcile the numerous and sometimes conflicting studies on the targeting of SHM to immunoglobulin loci, and highlight areas that hold promise for further investigation.     

Enhanced gene trapping in mouse embryonic stem cells

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.     

C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion

Class-switch recombination (CSR), somatic hypermutation (SHM), and antibody gene conversion are distinct DNA modification reactions, but all are initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. Here we describe a mutant form of AID that catalyzes SHM and gene conversion but not CSR. When expressed in E. coli, AID(delta189-198) is more active in catalyzing cytidine deamination than wild-type AID. AID(delta189-198) also promotes high levels of gene conversion and SHM when expressed in eukaryotic cells, but fails to induce CSR. These results underscore an essential role for the C-terminal domain of AID in CSR that is independent of its cytidine deaminase activity and that is not required for either gene conversion or SHM.     

人绒毛膜促性腺激素β亚基单一B-细胞表位(β5、β9或β8)融合蛋白的表达和纯化

作为开发新型实用性人绒毛膜促性腺激素(hCG)疫苗的一种尝试, 我们已构建若干组合靶抗原三个线性B- 细胞表位和外源强T- 细胞表位的基因工程hCG嵌合肽。为了检测用这些嵌合肽免疫的动物血清中是否能产生抗各表位的三种抗体,本研究选用能在大肠杆菌中高表达和与生物素亲和性强且特异(方便通过亲和层析纯化)的链霉亲和素为载体,分别构建了三种含β-hCG不同单一线性B_细胞表位(β5,β9和β8)的融合蛋白。在链霉亲和素基因下游多克隆区EcoRⅠ和Hind Ⅲ位点插入各表位编码基因片段(带TAA终止密码子)的pTSA-18重组质粒, 转化BL21(DE3)pLysS宿主菌后, 它们在IPTG诱导下均能以较高水平表达各自目的融合蛋白,而且它们的表达产物在Western blot鉴定中都能被抗各表位特异的多抗或单抗或抗报告表位单抗识别。用改良的制备性PAGE方法可以一步纯化电泳均一性高于95%的三个融合蛋白, 它们的收得率相对1L培养物约为5 mg。作为化学合成表位肽的替代物, β-hCG三个单一B- 细胞表位融合蛋白的可获得性将有助于所构建hCG基因工程嵌合肽以及其他hCG疫苗,也包括它的DNA疫苗的免疫原性分析。     

一种低温表达型T载体及其应用

在现代生物学和生物技术研究中,通过基因重组表达获得目标蛋白已成为常规技术。因其培养简单、操作方便、遗传背景清楚、克隆表达技术成熟,大肠杆菌表达系统通常是人们表达重组蛋白的首选。但是在常规温度下进行基因的重组表达,动、植物和常温微生物的基因产物多数在数小时内变性沉淀;还有一些重组蛋白对宿主具有细胞毒性,难以得到重组表达。因此,我们构建了1种新型T载体——pEXC-T;它结合TA克隆技术和低温诱导表达功能,具有表达水平高、操作方便、目标蛋白得到分子伴侣保护和低温保存等特点。采用构建和优化的pEXC载体,P1抗原蛋白、溶血素PLO两种不稳定性蛋白在pEXC中都实现了高效的可溶性表达。低温表达系统p EXC的建立和发展为蛋白质的结构与功能的研究,以及抗原和药用蛋白的制备提供了便利的途径。     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.