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P Engler

Publications and source records attributed to P Engler.

At least 19 recordsLinked to original sources
PubMed2001

An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection.

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.

Animals↗
PubMed1999

Hypomethylation is necessary but not sufficient for V(D)J recombination within a transgenic substrate.

Although an inverse correlation between CpG methylation and V(D)J recombination has been demonstrated for both artificial substrates and endogenous genes, it is not known whether all hypomethylated targets are competent to rearrange or if other factors are required. We have created several artificial V(D)J recombination substrate transgenes whose methylation can be controlled by breeding into different genetic backgrounds. A transgene which contains the immunoglobulin heavy chain intronic enhancer rearranges efficiently in B lymphocytes when the transgene loci are unmethylated. When the same loci become methylated, upon breeding into a different mouse strain, no rearrangement can be detected. A similar transgene, but lacking the enhancer, also shows no evidence of V(D)J recombination when it is methylated. Even when this enhancerless transgene is hypomethylated, however, no V(D)J recombination can be detected in B lymphocytes. Thus, hypomethylation is required to permit V(D)J recombination but not all hypomethylated targets are capable of recombination. The results may indicate that the immunoglobulin enhancer is required for the assembly of factors involved in V(D)J recombination.

Animals↗
PubMed1998

A cis-acting element that directs the activity of the murine methylation modifier locus Ssm1.

Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature of Ssm1's targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism of Ssm1 action are discussed.

Animals↗
PubMed1995

Asymmetric processing of coding ends and the effect of coding end nucleotide composition on V(D)J recombination.

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.

B-Lymphocytes↗
PubMed1995

The bulk chromatin structure of a murine transgene does not vary with its transcriptional or DNA methylation status.

The DNA methylation status of HRD, a murine transgene, can be controlled by the genetic background upon which it is carried. We found the transgene to be transcribed in competent tissues only when undermethylated. Chromatin structure over the transgene was assayed by nuclear accessibility with DNase I, MspI, and PstI. While the transgene was up to fivefold more resistant to MspI when methylated than when not methylated, we observed no such difference with DNase I or PstI. We suggest that methyl-CpG-binding proteins are responsible for the difference observed with MspI, but that the chromatin structures are otherwise similarly compacted. Methylation could, therefore, play a regulatory role in gene expression beyond that which can be accomplished by bulk chromatin structure alone.

5-Methylcytosine↗
PubMed1994

Ablation of neoplasia by direct current.

The application of low-voltage direct electrical current (DEC) has been studied in animals and humans for the ablation of anal condylomata, oesophageal cancer and Kaposi's sarcoma. Twenty milliamps of DEC passed through multiple 6 cm x 1 cm, flat-plate longitudinal electrodes into the squamous mucosa of the oesophagus of healthy dogs for periods ranging from 10 min to 2 h resulted in denudation and necrosis of the oesophageal mucosa at the site of application of the current. In humans, the application of DEC to two patients with benign anal condyloma acuminata, three patients with inoperable obstructing oesophageal cancer and one patient with disseminated Kaposi sarcoma resulted in striking necrosis of tumour tissue that was confirmed by macroscopic and microscopic studies. These initial findings imply promising therapeutic potential for the use of DEC as a simple, effective, safe, low-cost alternative for ablation of neoplasia.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Influence of CpG methylation and target spacing on V(D)J recombination in a transgenic substrate.

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.

5-Methylcytosine↗
PubMed1992

N region diversity of a transgenic substrate in fetal and adult lymphoid cells.

The rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes requires the activity of an as yet undefined V(D)J recombinase. One component of the recombinase appears to be a terminal transferase which may be involved in the addition of untemplated nucleotides (N regions) to the V(D)J joints. It has been observed that rearranged Ig and TCR genes isolated from fetal liver have few if any N regions, whereas in the adult mouse, these genes have a large number of untemplated nucleotides. The presence of N regions greatly alters the composition of the third hypervariable, complementarity determining region of the respective proteins, thus playing a major role in the conformation of the binding site. It was possible that, for functional reasons, N region-containing Ig and TCR genes were not permissible at the fetal stage of development. We have produced transgenic mice with a rearrangement test gene which, after V-J recombination, does not result in the production of functional Ig or TCR proteins. We report here that the rearrangement products have no N regions in fetal liver, but that the majority of joints in adult lymphoid tissues have N additions. The study is also an interesting demonstration of the randomness of rearrangements and the enormous variability that can be created from a single pair of V and J sequences.

Animals↗
PubMed1992

Rearrangement and expression of immunoglobulin genes in transgenic mice.

Transgenic mice are discussed which carry a rearrangement test transgene. The methylation status of the transgene varies, depending on the background mouse strain. When the transgene is bred into the C57BL/6 strain, it is completely methylated and not rearranged in lymphoid organs. After several generations of crossing into DBA/2 or SJL the transgene becomes unmethylated and rearranges at high frequency. A strain specific modifier of DNA methylation (Ssm-1) was mapped close to the Friend virus susceptibility locus (Fv-1) on mouse chromosome 4. Rearranged transgenes from spleen, bone marrow and thymus of adult mice or fetal liver were cloned and sequenced. A great variety of joints was found, with about 1/3 being in the correct reading frame. Small deletions into the V- and J-coding ends as well as N region additions contributed to the variability. The fetal joints showed no N regions. Since no functional immunoglobulin (Ig) gene can be created from this artificial test gene, the data indicate that the rearrangement mechanism of the fetus differs from that of the adult.

Animals↗
PubMed1991

A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci.

A transgene, pHRD, is highly methylated in 12 independent mouse lines when in a C57BL/6 strain background, but becomes progressively less methylated when bred into a DBA/2 background. Transgenes inherited from the mother are generally more methylated; however, this parental effect disappears following continued breeding into the nonmethylating strain. Mapping experiments using BXD recombinant inbred mice as well as other inbred strains indicate that a single strain-specific modifier (Ssm-1) linked to, but distinct from, Fv-1 is responsible for the strain effect. In addition to the methylated and unmethylated transgenic phenotypes, certain mice exhibit a partial methylation pattern that is a consequence of an unusual cellular mosaicism. The pHRD transgene, containing target sequences for the V(D)J recombinase, undergoes site-specific recombination only in lymphoid tissues. This V-J joining is restricted primarily to unmethylated transgene copies.

Animals↗
PubMed1991

Factors affecting the rearrangement efficiency of an Ig test gene.

A rearrangement test gene, pHRD, containing the mouse IgH enhancer and the metallothionein promoter, has previously been shown to rearrange efficiently after transfection into a pre-B cell line. Experiments are now reported that assess the requirements of the DNA substrate as well as of the transfected cells for efficient rearrangement. It was found that deletion of the metallothionein promoter or substitution of the IgH enhancer by the kappa enhancer did not affect rearrangement. However, deletion of the Ig enhancer reduced the efficiency. Transfection of pHRD into stable hybrids of pre-B cells and myeloma cells resulted in a high frequency of rearrangement only if certain myeloma chromosomes were lost. Furthermore, pHRD introduced into rearrangement incompetent myeloma cells upon subsequent cell fusion with pre-B cells was rearranged only very rarely and then apparently only immediately after cell fusion. Stable pre-B cell x myeloma hybrids that retained the critical myeloma chromosomes were found to have lost VDJ recombinase activity and transcripts of the RAG-1, RAG-2 and TdT genes. It is concluded that transcription, i.e., the copying of the DNA by polymerase, is probably not required for rearrangement, but that the rearrangement substrate must be in an "open" chromatin state, such as may be provided by transcriptional factors. Furthermore, the absence of rearrangement in myeloma cells is apparently due to the continued action of an inhibitor of rearrangement.

Animals↗
PubMed1990

Differential splicing of thymosin beta 4 mRNA.

A cDNA clone was isolated from a mouse pre-B cell line, the sequence of which has a very high homology with rat and human thymosin beta 4 genes. However, the mouse clone has an insertion of 98 bp relative to the published rat and human sequences upstream of the coding region. By isolation of a second set of clones from a different cDNA library and by cloning a PCR amplified region of mouse genomic DNA it was confirmed that the insertion is not a cloning artifact. Furthermore, it was shown by RNase protection assays with RNA from the pre-B cell line that two sizes of thymosin beta 4 mRNA exist, a long form containing the 98 nucleotide insertion, and a short form that corresponds to the known rat and human mRNA. The short form is about 50 times more abundant than the long form. Analysis of genomic DNA by sequencing and Southern blotting revealed that both forms are encoded by a single gene in the mouse. The two forms of mRNA arise by differential RNA splicing; the long mRNA contains three separate exons, whereas the short mRNA is missing exon 2. The long mRNA is present in two different pre-B cell lines, spleen and thymus, but could not be detected in brain, liver, and kidney. It is possible that the longer mRNA, which encodes a hydrophobic NH2-extension of six additional amino acids, plays a role in lymphocyte function or development. In contrast to the mouse which has a single thymosin beta 4 gene, rat and human have multiple homologs. Most or all of these also contain sequences that cross-hybridize with the newly discovered exon 2. A polymorphic thymosin beta 4 gene has been found in human DNA.

Amino Acid Sequence↗
PubMed1988

Expression of immunoglobulin genes in transgenic mice and transfected cells.

Immunoglobulin (Ig) genes are expressed sequentially (first H-, then L-chain genes) during the development of B lymphocytes. These studies, performed with transgenic mice and transfected cells, were aimed at the regulation of turning on and off the rearrangement of Ig genes. The specific recombinase is active in pre-B cells, but not in plasma cells. Production of membrane mu, but not secreted mu or gamma-2b, turns off rearrangement of H genes. Feedback inhibition of kappa-gene rearrangement requires kappa and membrane mu. Kappa alone or in combination with secreted mu does not stop recombination. Mouse lambda genes were mapped by deletion analysis and pulsed-field gel electrophoresis. The gene order is V2-C2,4-V1-C3,C1. The distance between V2 and C2 is 74 kb, but that between V1 and C3, 1 is only 20 kb. V2 and C3, 1 are over 190 kb apart. Lambda genes appear to be rearranged in a subset of B cells that do not respond to feedback inhibition at the pre-B cell stage. Lambda and kappa genes are both rearranged and potentially functional in these cells. Kappa genes may then be deleted by recombination of a sequence (described by Selsing and Siminovitch et al.) downstream of C-kappa with sequences upstream of C-kappa. Presumably the recombinase is eventually inactivated in kappa-lambda cells by a mechanism that is different from H-kappa feedback.

Animals↗
PubMed1987

High-frequency deletional rearrangement of immunoglobulin kappa gene segments introduced into a pre-B-cell line.

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 kappa chain variable region (V kappa) and from a kappa chain joining region (J kappa). 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, greater than 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; less than 45 base pairs 5' of the J nonamer and 3' of its heptamer) are required for efficient rearrangement and provide formal proof that kappa gene segments can rearrange by a deletional mechanism.

Antibody Diversity↗
PubMed1987

The effect of temperature on survival in hemorrhagic shock.

Hypothermia is a frequent complication of severe hemorrhagic shock and can complicate the treatment of trauma patients. The authors have investigated the effect of external warming on a treated model of hemorrhagic shock in rats. Their data show that externally heating the animal during the shock period decreases the animal's ability to withstand shock and increases intrashock and postshock mortality when compared to nonheated controls. The authors have also shown that nonheated animals that can retain body heat by passive retention can withstand increased shock time and have an increased postshock survival.

Animals↗
PubMed1986

Transgenic mice with mu and kappa genes encoding antiphosphorylcholine antibodies.

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.

Animals↗