We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL)... more We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10–50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZ...
The Sleeping Beauty (SB) transposon system represents a new vector for non-viral gene transfer th... more The Sleeping Beauty (SB) transposon system represents a new vector for non-viral gene transfer that melds advantages of viruses and other forms of naked DNA transfer. The transposon itself is comprised of two inverted terminal repeats of about 340 base pairs each. The SB system directs precise transfer of specific constructs from a donor plasmid into a mammalian chromosome. The excision of the transposon from a donor plasmid and integration into a chromosomal site is mediated by Sleeping Beauty transposase, which can be delivered to cells vita its gene or its mRNA. As a result of its integration in chromosomes, and its lack of viral sequences that are often detected by poorly understood cellular defense mechanisms, a gene in a chromosomally integrated transposon can be expressed over the lifetime of a cell. SB transposons integrate nearly randomly into chromosomes at TA-dinucleotide base pairs although the sequences flanking the TAs can influence the probability of integration at a given site. Although random integration of vectors into human genomes is often thought to raise significant safety issues, evidence to date does not indicate that random insertions of SB transposons represent risks that are equal to those of viral vectors. Here we review the activities of the SB system in mice used as a model for human gene therapy, methods of delivery of the SB system, and its efficacy in ameliorating disorders that model human disease.
... 3,4 , Jason B. Bell 2,4 , Roland Gunther 5 , Brenda Koniar 5 , Aric Frantz 6 , Ilze Matise 7 ... more ... 3,4 , Jason B. Bell 2,4 , Roland Gunther 5 , Brenda Koniar 5 , Aric Frantz 6 , Ilze Matise 7 , R. Scott McIvor 1 ... However, our attempts to achieve long-term expression of human alpha -L-iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in ...
Abstract Retroviruses and transposons are mobile genetic elements with the ability to integrate g... more Abstract Retroviruses and transposons are mobile genetic elements with the ability to integrate genetic information into chromosomes for human gene therapy. However, integration-site preferences for mobile elements are poorly understood. We analyzed the ...
Page 1. ANIMAL BIOTECHNOLOGY, 1(1), 79-93 (1990) GENE EXPRESSION PROMOTED BY THE RSV LONG TERMINA... more Page 1. ANIMAL BIOTECHNOLOGY, 1(1), 79-93 (1990) GENE EXPRESSION PROMOTED BY THE RSV LONG TERMINAL REPEAT ELEMENT IN TRANSGENIC GOLDFISH Eric M. Hallerman1,6, John F. Schneider2, Mark Gross3, Zhanjiang Liu4, ...
We have previously shown that human serum albumin (HSA) enhances PEI-mediated gene transfer into ... more We have previously shown that human serum albumin (HSA) enhances PEI-mediated gene transfer into immortalized cell lines derived from various anatomical regions of the airways (1). Therefore, we sought to confirm these results on ex-vivo cultures of primary respiratory cells: nasal polyps, nasal brushings and bronchial cells obtained from lung biopsies. The complexes were prepared by adding different amounts of HSA (0.2 -20 µg) to preformed PEI/DNA polyplexes. HSA increased PEI-mediated gene transfer in nasal outgrowths (n=5) and nasal cells obtained from brushings (n=3) by 2.6 and 4.8 times, respectively. Transfection of primary bronchial epithelial cells by HSA/PEI/DNA complexes was increased 3.2 times, relative to values obtained with PEI.
Background: Insertional mutagenesis techniques with transposable elements have been popular among... more Background: Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue-or temporal-specific pattern.
Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate re... more Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.
The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have bee... more The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.
Proceedings of the National Academy of Sciences, 1996
DANA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of thes... more DANA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms betweeit zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.
Proceedings of the National Academy of Sciences, 2002
Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic ... more Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA͞transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.
Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrat... more Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.
A study of the secondary structures of the 5'-leader RNA sequences of avian leukosis/sarcoma viru... more A study of the secondary structures of the 5'-leader RNA sequences of avian leukosis/sarcoma viruses was conducted using phylogenetic sequence alignment, theoretical structures calculated from base-pairing interactions involving the calculated minimal AG values, and RNaseTl sensitivitiy. The results suggest that all of the avian retroviral RNA leaders may be able to adopt similar conformations. Open reading frames in the leader RNAs may be positioned to facilitate viral activities such as translation and packaging of the genomic RNA into virus particles.
Mobile genetic elements with the ability to integrate genetic information into chromosomes can ca... more Mobile genetic elements with the ability to integrate genetic information into chromosomes can cause disease over short periods of time and shape genomes over eons. These elements can be used for functional genomics, gene transfer and human gene therapy. However, their integration-site preferences, which are critically important for these uses, are poorly understood. We analyzed the insertion sites of several transposons and retroviruses to detect patterns of integration that might be useful for prediction of preferred integration sites. Initially we found that a mathematical description of DNAdeformability, called V step , could be used to distinguish preferential integration sites for Sleeping Beauty (SB) transposons into a particular 100 bp region of a plasmid (2005) J. Mol. Biol., 346,[161][162][163][164][165][166][167][168][169][170][171][172][173]. Based on these findings, we extended our examination of integration of SB transposons into whole plasmids and chromosomal DNA. To accommodate sequences up to 3 Mb for these analyses, we developed an automated method, ProTIS Ó , that can generate profiles of predicted integration events. However, a similar approach did not reveal any structural pattern of DNA that could be used to predict favored integration sites for other transposons as well as retroviruses and lentiviruses due to a limitation of available data sets. Nonetheless, ProTIS Ó has the utility for predicting likely SB transposon integration sites in investigator-selected regions of genomes and our general strategy may be useful for other mobile elements once a sufficiently high density of sites in a single region are obtained. ProTIS analysis can be useful for functional genomic, gene transfer and human gene therapy applications using the SB system.
We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL)... more We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10–50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZ...
The Sleeping Beauty (SB) transposon system represents a new vector for non-viral gene transfer th... more The Sleeping Beauty (SB) transposon system represents a new vector for non-viral gene transfer that melds advantages of viruses and other forms of naked DNA transfer. The transposon itself is comprised of two inverted terminal repeats of about 340 base pairs each. The SB system directs precise transfer of specific constructs from a donor plasmid into a mammalian chromosome. The excision of the transposon from a donor plasmid and integration into a chromosomal site is mediated by Sleeping Beauty transposase, which can be delivered to cells vita its gene or its mRNA. As a result of its integration in chromosomes, and its lack of viral sequences that are often detected by poorly understood cellular defense mechanisms, a gene in a chromosomally integrated transposon can be expressed over the lifetime of a cell. SB transposons integrate nearly randomly into chromosomes at TA-dinucleotide base pairs although the sequences flanking the TAs can influence the probability of integration at a given site. Although random integration of vectors into human genomes is often thought to raise significant safety issues, evidence to date does not indicate that random insertions of SB transposons represent risks that are equal to those of viral vectors. Here we review the activities of the SB system in mice used as a model for human gene therapy, methods of delivery of the SB system, and its efficacy in ameliorating disorders that model human disease.
... 3,4 , Jason B. Bell 2,4 , Roland Gunther 5 , Brenda Koniar 5 , Aric Frantz 6 , Ilze Matise 7 ... more ... 3,4 , Jason B. Bell 2,4 , Roland Gunther 5 , Brenda Koniar 5 , Aric Frantz 6 , Ilze Matise 7 , R. Scott McIvor 1 ... However, our attempts to achieve long-term expression of human alpha -L-iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in ...
Abstract Retroviruses and transposons are mobile genetic elements with the ability to integrate g... more Abstract Retroviruses and transposons are mobile genetic elements with the ability to integrate genetic information into chromosomes for human gene therapy. However, integration-site preferences for mobile elements are poorly understood. We analyzed the ...
Page 1. ANIMAL BIOTECHNOLOGY, 1(1), 79-93 (1990) GENE EXPRESSION PROMOTED BY THE RSV LONG TERMINA... more Page 1. ANIMAL BIOTECHNOLOGY, 1(1), 79-93 (1990) GENE EXPRESSION PROMOTED BY THE RSV LONG TERMINAL REPEAT ELEMENT IN TRANSGENIC GOLDFISH Eric M. Hallerman1,6, John F. Schneider2, Mark Gross3, Zhanjiang Liu4, ...
We have previously shown that human serum albumin (HSA) enhances PEI-mediated gene transfer into ... more We have previously shown that human serum albumin (HSA) enhances PEI-mediated gene transfer into immortalized cell lines derived from various anatomical regions of the airways (1). Therefore, we sought to confirm these results on ex-vivo cultures of primary respiratory cells: nasal polyps, nasal brushings and bronchial cells obtained from lung biopsies. The complexes were prepared by adding different amounts of HSA (0.2 -20 µg) to preformed PEI/DNA polyplexes. HSA increased PEI-mediated gene transfer in nasal outgrowths (n=5) and nasal cells obtained from brushings (n=3) by 2.6 and 4.8 times, respectively. Transfection of primary bronchial epithelial cells by HSA/PEI/DNA complexes was increased 3.2 times, relative to values obtained with PEI.
Background: Insertional mutagenesis techniques with transposable elements have been popular among... more Background: Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue-or temporal-specific pattern.
Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate re... more Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.
The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have bee... more The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.
Proceedings of the National Academy of Sciences, 1996
DANA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of thes... more DANA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms betweeit zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.
Proceedings of the National Academy of Sciences, 2002
Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic ... more Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA͞transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.
Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrat... more Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.
A study of the secondary structures of the 5'-leader RNA sequences of avian leukosis/sarcoma viru... more A study of the secondary structures of the 5'-leader RNA sequences of avian leukosis/sarcoma viruses was conducted using phylogenetic sequence alignment, theoretical structures calculated from base-pairing interactions involving the calculated minimal AG values, and RNaseTl sensitivitiy. The results suggest that all of the avian retroviral RNA leaders may be able to adopt similar conformations. Open reading frames in the leader RNAs may be positioned to facilitate viral activities such as translation and packaging of the genomic RNA into virus particles.
Mobile genetic elements with the ability to integrate genetic information into chromosomes can ca... more Mobile genetic elements with the ability to integrate genetic information into chromosomes can cause disease over short periods of time and shape genomes over eons. These elements can be used for functional genomics, gene transfer and human gene therapy. However, their integration-site preferences, which are critically important for these uses, are poorly understood. We analyzed the insertion sites of several transposons and retroviruses to detect patterns of integration that might be useful for prediction of preferred integration sites. Initially we found that a mathematical description of DNAdeformability, called V step , could be used to distinguish preferential integration sites for Sleeping Beauty (SB) transposons into a particular 100 bp region of a plasmid (2005) J. Mol. Biol., 346,[161][162][163][164][165][166][167][168][169][170][171][172][173]. Based on these findings, we extended our examination of integration of SB transposons into whole plasmids and chromosomal DNA. To accommodate sequences up to 3 Mb for these analyses, we developed an automated method, ProTIS Ó , that can generate profiles of predicted integration events. However, a similar approach did not reveal any structural pattern of DNA that could be used to predict favored integration sites for other transposons as well as retroviruses and lentiviruses due to a limitation of available data sets. Nonetheless, ProTIS Ó has the utility for predicting likely SB transposon integration sites in investigator-selected regions of genomes and our general strategy may be useful for other mobile elements once a sufficiently high density of sites in a single region are obtained. ProTIS analysis can be useful for functional genomic, gene transfer and human gene therapy applications using the SB system.
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Papers by Perry Hackett