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Transgenic mice were produced that carried in their germlines rearranged kappa and/or mu genes with V kappa and VH regions from the myeloma MOPC-167 kappa and H genes, which encode anti-PC antibody. The mu genes contain either a complete gene, including the membrane terminus (mu genes), or genes in which this terminus is deleted and only the secreted terminus remains (mu delta mem genes). The mu gene without membrane terminus is expressed at as high a level as the mu gene with the complete 3' end, suggesting that this terminus is not required for chromatin activation of the mu locus or for stability of the mRNA. The transgenes are expressed only in lymphoid organs. In contrast to our previous studies with MOPC-21 kappa transgenic mice, the mu transgene is transcribed in T lymphocytes as well as B lymphocytes. Thymocytes from mu and kappa mu transgenic mice display elevated levels of M-167 mu RNA and do not show elevated levels of kappa RNA, even though higher than normal levels of M-167 kappa RNA are detected in the spleen of these mice. Approximately 60% of thymocytes of mu transgenic mice produce cytoplasmic mu protein. However, despite a large amount of mu RNA of the membrane form, mu protein cannot be detected on the surface of T cells, perhaps because it cannot associate with T cell receptor alpha or beta chains. Mice with the complete mu transgene produce not only the mu transgenic mRNA but also considerably increased amounts of kappa RNA encoded by endogenous MOPC-167 like kappa genes. This suggests that B cells are selected by antigen (PC) if they coexpress the mu transgene and appropriate anti-PC endogenous kappa genes. Mice with the mu delta mem gene, however, do not express detectable levels of the endogenous MOPC-167 kappa mRNA. Like the complete mu transgene, the M-167 kappa transgene also causes amplification of endogenous MOPC-167 related immunoglobulins; mice with the kappa transgene have increased amounts of endogenous MOPC-167-like mu or alpha or gamma in the spleen, all of the secreted form. Implications for the regulation of immunoglobulin gene expression and B cell triggering are discussed.

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BACs in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs), with their capacity for large inserts, stability, and lack of chimerism, play important roles in genome mapping and sequencing (Osoegawa et al. 2000). For these reasons and the fact that virtually every gene will have one or more corresponding BAC/PAC clones, they are the first choice for functional complementation or dose-effect studies in differentiated or embryonic stem (ES) cells. Mouse ES cells are of special interest for gene function studies because of their capacity for in vitro differentiation into different lineages (O'Shea 1999) and the ability to generate transgenic animals through germline transmission. The lack of a universal selection marker for mammalian cell transfection makes it difficult to include any particular marker in the BAC/PAC vectors. Therefore, the vectors for most current BAC or PAC libraries do not contain any mammalian selection marker for transfection studies. This necessitates the development of efficient ways to insert such selection markers into individual clones chosen from BAC/PAC libraries, a "retrofitting" operation.Several strategies have been developed to retrofit BACs/PACs on the basis of restriction digestion, transposition, homologous recombination, or site-specific recombination. Certain rare-cutter restriction enzymes have been used to linearize both the BAC clone and a retrofitting cassette. These sticky ends were then ligated to form retrofitted BACs with the selectable marker inserted (Mejia and Monaco 1997;Hejna et al. 1998). For this strategy to work, it has to be certain that there is no such restriction site in the genomic insert, which often is not the case with large inserts. It also requires subsequent transformation of the retrofitted BAC/PAC into Escherichia coli, which is both very inefficient and deleterious to the genomic insert. Recombination-based approaches allow the direct...

We have previously described a line of transgenic mice with multiple head-to-tail copies of an artificial V-J recombination substrate and have shown that the methylation of this transgene is under the control of a dominant strain-specific modifier gene, Ssm-1. When the transgene array is highly methylated, no recombination is detectable, but when it is unmethylated, V-J joining is seen in the spleen, bone marrow, lymph nodes, and Peyer's patches but not in the thymus or nonlymphoid tissues, including brain tissue. Strikingly, in mice with partially methylated transgene arrays, rearrangement preferentially occurs in hypomethylated copies.Therefore, V-J recombination is negatively correlated with methylated DNA sequences. In addition, it appears that recombination occurs randomly between any two recombination signal sequences within the transgene array. This lack of target preference in an unselectable array of identical targets rules out simple mechanisms of one-dimensional tracking of a V(D)J recombinase complex.During lymphoid development, antigen receptor genes are assembled from separate coding elements. There are three requirements for this site-specific DNA joining: recombination signal sequences (RSSs), V(D)J recombinase, and accessibility, which allows productive interaction between the recombinase and its targets.RSSs consisting of a conserved heptamer and a nonamer separated by spacers of either 12 or 23 bp are found adjacent to all coding elements. Studies with artificial substrates have demonstrated that these are the only sequence elements necessary for V(D)J recombination (2), suggesting that these signal sequences are recognized by the recombinase. Extensive mutational analysis (17) of these elements has shown that the consensus sequences, CACAGTG and ACAAAA ACC, function most efficiently, although substantial variation is tolerated particularly in the nonamer and the codingdistal half of the heptamer. Recombination almost invariably occurs between partners having RSSs with different spacer lengths (the "one turn/two turn" rule).The V(D)J recombinase is less well understood, but there is evidence for the involvement of a number of genes and/or proteins. V(D)J recombination appears to be restricted to lymphoid cells (but see reference 23 for an alternative view). However, this ability can be conferred upon other cell types by the forced expression of Rag-i and Rag-2 (27, 30). These genes are normally expressed together only in early lymphoid cells, and their expression closely parallels V(D)J recombinase activity, suggesting that they encode central components of the recombinase. Direct biochemical evidence for this is lacking so far. Evidence that numerous other components might be involved in V(D)J recombination has been presented (reviewed in reference 22).The molecular nature of accessibility is poorly understood. All V(D)J recombination seems to be carried out by a common recombinase (34), yet B cells rearrange immunoglobulin (Ig), but not T-cell receptor (TCR), loci, while the converse is gene...

The products of V(D)J recombination are coding and signal joints. We show that the nucleotide composition of the coding ends affects V(D)J recombination. The presence of Ts at the 5' end of either the 12 mer or the 23 mer recombination signal sequence (RSS) greatly decreases coding and signal joint formation, and Ts at the 5' ends of both RSSs eliminate recombination, suggesting that a step during the initiation phase of the recombination is affected. A 5' T coding end can be rescued it the other end contains 5' G, C, or A, implying that synapsis may be required. Furthermore, the presence of As at the 5' end of the 12 mer, but not the 23 mer, RSS affects coding but not signal joint formation. This observation of asymmetric processing of coding ends suggests that different protein complexes are bound to the two RSSs, and become transferred to the aligned coding ends during processing.

The strain-specific modifier Ssm1 is responsible for the straindependent methylation of particular E. coli gpt-containing transgenic sequences. Here, we identify Ssm1 as the KRAB-zinc finger (ZF) gene 2610305D13Rik located on distal chromosome 4. Ssm1b is a member of a gene family with an unusual array of three ZFs. Ssm1 family members in C57BL/6 (B6) and DBA/2 (D2) mice have various amino acid changes in their ZF domain and in the linker between the KRAB and ZF domains. Ssm1b is expressed up to E8.5; its target transgene gains partial methylation by this stage as well. At E9.5, Ssm1b mRNA is no longer expressed but by then its target has become completely methylated. By contrast, in D2 embryos the transgene is essentially unmethylated. Methylation during B6 embryonic development depends on Dnmt3b but not Mecp2. In differentiating B6 embryonic stem cells methylation spreads from gpt to a co-integrated neo gene that has a similarly high CpG content as gpt, but neo alone is not methylated. In adult B6 mice, Ssm1b is expressed in ovaries, but in other organs only other members of the Ssm1 family are expressed. Interestingly, the transgene becomes methylated when crossed into some, but not other, wild mice that were kept outbred in the laboratory. Thus, polymorphisms for the methylation patterns seen among laboratory inbred strains are also found in a free-living population. This may imply that mice that do not have the Ssm1b gene may use another member of the Ssm1 family to control the potentially harmful expression of certain endogenous or exogenous genes.

We describe an immunoglobulin gene recombination indicator in which a specific rearrangement via deletion results in the acquisition of a dominant phenotype. The indicator consists of the Escherichia coli xanthine/guanine phosphoribosyltransferase (gpt) gene, whose translation is prevented by the presence of an upstream initiation codon out of frame with respect to the gpt coding sequence. Flanking this barrier initiation codon are the heptamer-spacer-nonamer recognition sequences from a K chain variable region (Va) and from a K chain joining region (Jo). A proper V-J joint results in the deletion of the translational barrier and allows expression of the selectable marker. When tested by transfection into fibroblasts, no rearrangements were detected and the presence of the barrier initiation codon was sufficient to completely abolish gpt expression in these cells. Similarly, no rearrangements were detected after transfer of the test gene into myeloma cells. However, when the construct was introduced into the pre-B-cell line 38B9, >80% of the transfected cells showed evidence of a specific rearrangement. These rearrangements were associated with the translation of gpt, although no selection for its expression was needed. DNA sequence analysis of six different V-J joints revealed that the rearrangement proceeded with a high degree of accuracy. These results indicate that only very minimal DNA sequences (21 base pairs 5' of the V heptamer and 4 base pairs 3' of its nonamer; <45 base pairs 5' of the J nonamer and 3' of its heptamer) are required for efficient rearrangement and provide formal proof that K gene segments can rearrange by a deletional mechanism.As postulated over 20 years ago, the variable (V) and constant regions of immunoglobulin chains are encoded by separate genetic elements (1). During the development of an antibody-producing cell, these elements are rearranged to yield functional immunoglobulin genes. Closely associated with each of these elements [V, D (diversity), and J (joining) in the case of heavy-chain genes; V and J for light-chain genes; see ref. 2 for a review] are highly conserved DNA sequences consisting ofa heptamer, a spacer, and a nonamer, which presumably constitute at least a portion of the sites recognized by the enzyme(s) involved in the recombination.Much of our knowledge of the process of immunoglobulin gene rearrangement has come from comparison of the germline genes with their rearranged counterparts. For a more thorough understanding of various aspects of the recombination, a system that actively rearranges introduced substrates is desirable. Recently, two such systems in which recombination substrates are introduced into pre-B-cell lines have been described and have contributed greatly to our understanding of immunoglobulin gene rearrangement. One system depends on a VK-JK rearrangement via inversion to flip a selectable marker gene into the proper transcriptional orientation (3, 4). A number of recombinants have been isolated and sequenced, revealing that authentic...

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