Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs

Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection‐mediated gene transfer (ICSI‐MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI‐MGT yielded significantly more embryos with non‐mosaic transgene expression (P < 0.01). In the ICSI‐MGT group, 61.5% (24/39) of the embryos were EGFP‐positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP‐positive in the PN‐injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI‐MGT was transgenic, suggesting that ICSI‐MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI‐MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple‐copy, single‐site integration of a transgene is the primary outcome of ICSI‐MGT in the pig and that ICSI‐MGT is less likely than PN injection to cause transgene integration in a mosaic manner. Mol. Reprod. Dev. 79: 218–228, 2012. © 2011 Wiley Periodicals, Inc.     

A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting.

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.     

Association of transgene integration sites with chromosome rearrangements in hexaploid oat

Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombardment were characterized using phenotypic and genotypic segregation, Southern blot analysis, and fluorescence in situ hybridization (FISH). Twenty-five transgene loci were detected; 8 lines exhibited single transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A/D-genome-specific dispersed and clustered repeats showed no preferences in the distribution of transgene loci among the highly heterochromatic C genome and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes. Transgene integration sites were detected at different locations along individual chromosomes, although the majority of transformants had transgenes integrated into subtelomeric and telomeric regions. Transgene integration sites exhibited different levels of structural complexity, ranging from simple integration structures of two apparently contiguous transgene copies to tightly linked clusters of multiple copies of transgenes interspersed with oat DNA. The size of the genomic interspersions observed in these transgene clusters was estimated from FISH results on prometaphase chromosomes to be megabases long, indicating that some transgene loci were significantly larger than previously determined by Southern blot analysis. Overall, 6 of the 25 transgene loci were associated with rearranged chromosomes. These results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal breakage and rearrangements. Received: 29 July 1999 / Accepted: 9 November 1999     

Transgene copy number-dependent rescue of murine beta-globin knockout mice carrying a 183 kb human beta-globin BAC genomic fragment

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.     

Elucidation of the minimal sequence required to imprint H19 transgenes

The imprinted mouse H19 gene exhibits maternal allele-specific expression and paternal allele-specific hypermethylation. We previously demonstrated that a 14-kb H19 minitransgene possessing 5' differentially methylated sequence recapitulates the endogenous H19 imprinting pattern when present as high-copy arrays. To investigate the minimal sequences that are sufficient for H19 transgene imprinting, we have tested new transgenes in mice. While transgenes harboring limited or no 3' H19 sequence indicate that multiple elements within the 8-kb 3' fragment are required for appropriate imprinting, transgenes incorporating 1.7 kb of additional 5' sequence mimic the endogenous H19 pattern, including proper imprinting of low-copy arrays. One of these imprinted lines had a single 15.7-kb transgene integrant. This is the smallest H19 transgene identified thus far to display imprinting properties characteristic of the endogenous gene, suggesting that all cis-acting elements required for H19 imprinting in endodermal tissues reside within the 15.7-kb transgenic sequence.     

Xenopus laevis transgenesis by sperm nuclear injection

The stable integration of transgenes into embryos of the frog Xenopus laevis is achieved using the procedure described here. Linear DNA containing the transgene is incorporated randomly into sperm nuclei that have had their membranes disrupted with detergent treatment. Microinjection of these nuclei into unfertilized eggs produces viable embryos that can be screened for activity of the transgene. The proportion of embryos that harbor the transgene varies from 10 to 40% of the total number of surviving embryos. Multiple copies of the transgene can integrate as a concatemer into the sperm genome, and more than one site of DNA integration might occur within resulting animals. Germ cell transmission of the transgene is routine and the procedure is well suited to the production of transgenic reporter frog lines. One day should be allocated for the preparation of the sperm nuclei, which are stored as aliquots for future use. The transgenesis reaction and egg injection take one morning.     

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley ( Hordeum vulgare L.)

Introduced transgenes, uidA, sgfp (S65T) and/or bar, were localized using fluorescence in situ hybridization (FISH) on metaphase chromosomes of transgenic barley produced by microparticle bombardment of immature embryos. Of the 19 independent transgenic lines (eight diploid and 11 tetraploid), nine had uidA and ten had s gfp (S65T). All lines tested had three or more copies of the transgenes and 18 out of 19 lines had visibly different integration sites. At a gross level, it appeared that no preferential integration sites of foreign DNA among chromosomes were present in the lines tested; however, a distal preference for transgene integration was observed within the chromosome. In diploid T0 plants that gave a 3:1 segregation ratio of transgene expression in the T1, only single integration sites were detected on one of the homologous chromosomes. Homozygous diploid plants had doublet signals on a pair of homologous chromosomes. All tetraploid T0 plants that gave a 3:1 segregation ratio in the T1 generation had only a single integration site on one of the homologous chromosomes. In contrast, the single tetraploid T0 plant with a 35:1 segregation ratio in the T1 generation had doublet signals on a pair of homologous chromosomes. In the one tetraploid T0 line, which had a homozygote-like segregation ratio (45:0), there were doublet signals at two loci on separate chromosomes. We conclude that the application of FISH for analysis of transgenic plants is useful for the gross localization of transgene(s) and for early screening of homozygous plants.     

Rapid localization of transgenes in mouse chromosomes with a combined Spectral Karyotyping/FISH technique

We explored the feasibility of combined Spectral Karyotyping (SKY) and Fluorescence In Situ Hybridization (FISH) as means to rapidly map a chromosomally integrated renin/green fluorescent protein (GFP) fusion gene construct (Ren-GFP) in the transgenic mouse, Tg(Ren-GFP)1Kwg. A sequential hybridization with SKY probes followed by FISH gave consistently satisfactory results, demonstrating that multiple copies of the Ren-GFP transgene in this transgenic mouse line are integrated into a single chromosomal site of Chromosome (Chr) 4, most probably in the juxta-centromeric euchromatic region consisting of the A2-A3 domain. Chr 4 as a sole carrier of the transgene also was confirmed by co-hybridization to p1 BAC clone DNA containing telomeric sequences specific for mouse Chr 4 and the Ren-GFP construct in pGEM4Z vector. The hemizygosity of the Ren-GFP transgene is maintained not only in bone marrow cells, but also in lung cells proliferating in vitro, indicating that stable integration of the Ren-GFP transgene into chromosomal DNA was established at a very early embryonic stage. We conclude that the SKY/FISH technique is a reliable and facile method for establishing the integration site of a transgene. As such, this protocol has obvious advantages over traditional backcross methods in terms of time, cost and labor for determining the chromosomal location of transgenes.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation

Expression of the mouse whey acidic protein (WAP) gene is specific to the mammary gland, is induced several thousand-fold during pregnancy, and is under the control of steroid and peptide hormones. To study developmental regulation of the mouse WAP gene, a 7.2-kilobase (kb) WAP transgene, including 2.6 kb of 5'- and 1.6 kb of 3'-flanking sequences, was introduced into mice. Of the 13 lines of mice examined, 6 expressed the transgenes during lactation at levels between 3 and 54% of the endogenous gene. Although expression was dependent on the site of integration, the transgenes within a given locus were expressed in a copy number-dependent manner and were coordinately regulated. The WAP transgenes were expressed specifically in the mammary gland, but showed a deregulated pattern of expression during mammary development. In all six lines of mice, induction of the WAP transgenes during pregnancy preceded that of the endogenous gene. During lactation, expression in two lines increased coordinately with the endogenous gene, and in three other lines of mice, transgene expression decreased to a basal level. These data indicate that the 7.2-kb gene contains some but not all of the elements necessary for correct developmental regulation. At a functional level it appears as if a repressor element, which inactivates the endogenous gene until late pregnancy, and an element necessary for induction during lactation are absent from the transgene. Complementary results from developmental and hormone induction studies suggest that WAP gene expression during pregnancy and lactation is mediated by different mechanisms.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Somatic hypermutation of immunoglobulin kappa may depend on sequences 3'' of C kappa and occurs on passenger transgenes.

We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism.     

A Transgene-Induced Mitotic Arrest Mutation in the Mouse Allelic with Oligosyndactylism

Oligosyndactylism (Os) is a radiation-induced mutation on mouse chromosome 8 associated with early postimplantation lethality in homozygotes and abnormal development of the limbs and kidneys in heterozygotes. The recessive lethal effect of Os is due to a mitotic block of the embryonic cells that becomes apparent at the blastocyst stage, but it is not known if the heterozygous effect of Os is due to haploinsufficiency of the gene responsible for the mitotic arrest, or is due to mutation(s) of other gene(s). We have recently described a transgene-induced recessive mutation, 94-A/K, that results in early postimplantation death of the embryos, and we have mapped this mutation to the same region of chromosome 8 where Os has been assigned. On the basis of complementation tests between transgenic and Os/+ mice, in vitro growth characteristics and increased mitotic index of 94-A/K embryos, and molecular structural analysis of 94-A and 94-K transgenic and Os/+ mice, we conclude that the 94-A/K mutation represents a new allele of Os. This insertional mutation should facilitate the isolation of a mammalian gene essential for normal progression of the cell cycle beyond metaphase.     

Effects of genomic position and copy number of Acyl-ACP thioesterase transgenes on the level of the target fatty acids in Brassica napus L.

The effects of genomic position and copy number of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes on the major target fatty acid, either lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, in transgenic Brassica napus seed oil were investigated. Four transgenic parental lines, transformed individually with the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1) and Cuphea-TE (Ch FatB1) transgenes, were crossed with the non-transgenic recipient genotypes '212/86' or 'QO4'. Bay-TE and Cuphea-TE F₁ seeds, which carry half the number of the construct copies compared to the self-pollinated seeds of the transgenic parents, showed significantly lower levels of the target fatty acid. Doubled haploid (DH) lines were developed through microspore culture from F₁ hybrids with the elm-TE or the Cuphea-TE transgenes. DH lines carrying one to five copies of the Cuphea-TE transgene displayed a positive correlation between transgene copy number and the target fatty acid C16:0 level (r = 0.77**). DH lines with elm-TE transgene copies at four different loci showed different C16:0 levels, with one of the loci (E-II) leading to significantly higher C16:0 levels. This study supports the importance of the selection of high transgene copy number and/or the optimum genomic integration site in order to achieve maximum expression levels of the target fatty acid in transgenic oil quality modification.     

Chromosome integration of BAC (bacterial artificial chromosome): evidence of multiple rearrangements

This paper reports our attempts to characterize transgene integration sites in transgenic mouse lines generated by the microinjection of large (from 30 to 145 kb) pig DNA fragments encompassing a mammary specific gene, the whey acidic protein gene (WAP). Among the various methods used, the thermal asymmetric interlaced (TAIL-) PCR method allowed us (1) to analyze transgene/genomic borders and internal concatamer junctions for eleven transgenic lines, (2) to obtain sequence information for seven borders, (3) to place three transgenes in the mouse genome, and (4) to obtain sequence data for seven transgene junctions in concatamers. Finally, we characterized various rearrangements in the borders and the inner parts of the transgene. The possibility of such complex rearrangements should be carefully considered when transgenic animals are produced with large genomic DNA fragments.     

Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.