Hepatocarcinogenesis (Z#2)/mutagenesis during initiation stage.

Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.

Creating seamless junctions independent of restriction sites in PCR cloning.

A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyldeoxycytosine (m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.

Hepatocarcinogenesis is the sequel to hepatitis in Z#2 alpha 1-antitrypsin transgenic mice: histopathological and DNA ploidy studies.

Z mutant-associated alpha 1-antitrypsin deficiency in human beings leads to hepatitis and, in some cases, hepatocellular carcinoma. To begin to delineate the molecular basis for the development of hepatocellular carcinoma in alpha 1-antitrypsin deficiency, we previously developed transgenic mice using human alpha 1-antitrypsin M and Z genomic clones. High-copy Z lineage mice (12 gene copies/haploid mouse genome; "Z#2") had hepatocytes distended with human alpha 1-antitrypsin deficiency globules. Hepatitis was present, and the morphological changes mimicked those observed in human alpha 1-antitrypsin deficiency-related liver disease. The numbers of hepatocytes containing alpha 1-antitrypsin globules decreased with age, and alpha 1-antitrypsin-negative nodular aggregates of hepatocytes increased in number and size. Hepatocytic dysplasia occurred as early as 6 wk and was almost universally present at 1 yr. Nodules of dysplastic cells demonstrating aneuploidy were seen as early as 10 wks. These became persistent, proliferative lesions. Dysplasia and aneuploidy distinctly increased with time and advancing microscopic stage as lesions progressed to malignancy. Tumors were seen after 1 yr as adenomas, which are aneuploid and most likely well-differentiated hepatocellular carcinoma, and borderline malignant lesions; and, in 82% of Z#2 mice 16 to 20 mo old, as invasive hepatocellular carcinoma. These observations suggest but do not conclusively prove that hepatocellular carcinoma in alpha 1-antitrypsin deficiency and other hepatic disorders arises as a result of a common, endogenously stimulated pathway for hepatocellular carcinogenesis.

Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage.

We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP. The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision. In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage. When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles. We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries. Approximately 10-100-fold enrichment of specific clones was observed after each panning round. The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery.

A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage.

We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.

Expression of a heterodimeric Fab antibody protein in one cloning step.

A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.

Bacteriophage cloning and Escherichia coli expression of a human IgM Fab.

We have combined the molecular biology methods of the polymerase chain reaction and recombinant DNA cloning in bacteriophage lambda to express a human IgM Fab in Escherichia coli using genes derived from an Epstein-Barr virus transformed cell line. This method comprises three cDNA amplifications and a single cloning step, culminating in the stable overexpression of mammalian heterodimeric recombinant protein in a prokaryotic host.

High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.

A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.

Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.

Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo[a]pyrene (B[a]P), or cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.

Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector.

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.

The use of transgenic mice for short-term, in vivo mutagenicity testing.

In order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacZ target gene. Following exposure to mutagens, this target can be rescued efficiently from genomic DNA prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus Escherichia coli plating cultures. Mutations in the target gene appear as colorless plaques on a background of blue plaques when plated on indicator agar. Spontaneous background levels were approximately 1 x 10(-5) in each of three mouse lineages analyzed. Exposure of lambda transgenic mice to N-ethyl-N-nitrosourea resulted in as much as a 14-fold induction in detected mutations over background levels. The assay is currently being modified to incorporate lacI as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.

Histopathology of alpha 1-antitrypsin liver disease in a transgenic mouse model.

Transgenic mice were constructed using human alpha 1-antitrypsin M and Z genomic clones. Livers of the M lineage mice showed slight cellular pleomorphism and immunohistochemically demonstrable finely granular alpha 1-antitrypsin material in hepatocytes. Z lineage mice with five gene copies per haploid mouse genome (Z#1) demonstrated fine granular alpha 1-antitrypsin material and a few large globules. In contrast, Z lineage mice with 12 gene copies per haploid mouse genome (Z#2) demonstrated hepatocytes filled with homogeneous, eosinophilic globules that were strongly reactive with diastase and periodic acid-Schiff and antibody to alpha 1-antitrypsin. Scattered microscopic polymorphonuclear leukocyte accumulations were seen that contained extracellular alpha 1-antitrypsin material, but there was neither histological nor serological evidence of mouse infectious hepatitis. In young animals, small clusters of hepatocytes lacking alpha 1-antitrypsin material were seen. These cells were the dominant population in older animals and formed nodular arrangements. Fibrosis was not demonstrable in neonatal and young animals or in any of the controls, but perisinusoidal fibrosis was seen in older Z#2 mice. Groups of hepatocytes without alpha 1-antitrypsin material showed dysplastic changes. We conclude that the transgenic mouse is a reliable and useful model in which to study the effects of alpha 1-antitrypsin in the liver because it demonstrates changes similar to those in the human disease.

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.

ExoMeth sequencing of DNA: eliminating the need for subcloning and oligonucleotide primers.

A method is reported for sequencing DNA based on exonuclease III digestion and strand protection by using modified nucleoside triphosphates. Up to 10 kilobases of sequence information may be obtained from each strand of a given template without subcloning. Prior knowledge of the restriction map is not important; prior knowledge of any of the sequence is not required. Nor are oligonucleotide primers needed. Double-stranded cosmids, plasmids, lambda phage, or linear molecules (including amplified molecules) may be used as starting material. The method creates a single-stranded template from these starting molecules, thus generating high-quality sequence ladders. Most commonly used DNA polymerases may be utilized, including reverse transcriptase and T7 DNA polymerase. The approach is "ordered", so little time is wasted on redundant sequencing.

Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library.

Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library. Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule. This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.

Neonatal growth delay in alpha-1-antitrypsin disease. Influence of genetic background.

We have developed a transgenic mouse strain, Z#2, which represents a model for alpha 1-protease inhibitor (alpha 1-antitrypsin: alpha 1-Pi)-associated liver disease (Dycaico et al., 1988). Fifteen percent of human infants with alpha 1-Pi disease develop non-viral hepatitis which is sometimes associated with growth retardation. Such hepatitis and growth retardation tend to occur in a subset of families with other alpha 1-Pi affected members who have had non-viral hepatitis. The Z#2 mouse strain exhibits non-viral hepatitis and growth retardation. This phenotype is more pronounced in transgenic offspring of crosses between Z#2 mice and DBA/2J inbred mice, and less pronounced in transgenic offspring of crosses between Z#2 and CBA/J inbred mice. Such phenotypic differences resemble the phenotypic differences seen in human families with alpha 1-Pi-associated liver disease.

Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties.

A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(-), contained within the vector, can be excised by f1 or M13 helper phage. The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the f1 bacteriophage origin of replication (1). Six of 21 restriction sites in the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.