Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.     

Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22 584–22 588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2 kb genomic DNA fragment 5′ of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, and β-geo hybrid reporter gene. The β-galactosidase (βGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, βGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial–temporal expression patterns of the βGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong βGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, βGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2 kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of βGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.     

Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes

Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer.     

A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field

A transgenic mouse line harbouring a β-galacdosidase reporter gene controlled by the proximal 2 kb promoter of Hoxa3 was previously generated to investigate the regulatory cues governing Hoxa3 expression in the mouse. Examination of transgenic embryos from embryonic day (E) 8.0 to E15.5 revealed regionally restricted reporter activity in the developing heart. Indeed, transgene expression specifically delineated cells from three distinct lineages: a subpopulation of the second heart field contributing to outflow tract myocardium, the cardiac neural crest cells and the pharyngeal endoderm. Manipulation of the Retinoic Acid (RA) signaling pathway showed that RA is required for correct expression of the transgene. Therefore, this transgenic line may serve as a cardiosensor line of particular interest for further analysis of outflow tract development.     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken β-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos. Mol. Reprod. Dev. 49:368–373, 1998. © 1998 Wiley-Liss, Inc.     

Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element

A plasmid containing the adenovirus E2 gene, a gene normally requiring E1A-mediated induction during viral infection, is expressed very poorly upon transfection into mouse L cells. If the same plasmid is transfected into 293 cells, which constitutively express the adenovirus E1A gene, or into L cells together with a plasmid containing the E1A gene, the E2 gene is expressed at higher levels. Cotransfection of the E2 plasmid with a plasmid containing the pseudorabies virus (a herpesvirus) immediate early gene results in an even higher increase in the level of E2 expression. In addition, efficient E2 expression in the absence of trans induction was obtained by inserting E1A upstream promoter sequences at the 5' or 3' end of the E2 gene, indicating that these E1A sequences possess enhancer properties. Thus the efficient expression of the E2 gene can be obtained either by a structural change in the gene itself or by a trans-acting induction.     

Transient expression of a human β-actin promoter/lacZ gene introduced into mouse embryos correlates with a low degree of methylation

We have analysed the correlation between expression and methylation for the human β-actin promoter introduced into mouse embryos. The β-actin promoter was fused to the reporter gene lacZ, and expression was analysed after pronuclear injection into fertilized mouse eggs. We analysed transient expression in in vitro cultured preimplantation embryos and expression after chromosomal integration in 5 independent lines of transgenic mice. The in vitro cultured preimplantation embryos expressed lacZ from the 2-cell to the blastocyst stages, and most abundantly at the morula stage. By increasing the amount of injected DNA, a larger proportion of embryos expressed lacZ. Embryos expressing lacZ in only a subset of the blastomeres were detected at all preimplantation stages. In contrast to the transient expression after injection, we have not detected lacZ expression in any of the 5 analysed lines of transgenic mice carrying the same construct. The lack of expression in transgenic mice correlates with hypermethylation of C residues in the vast majority of CG sequences in the integrated β-actin/lacZ construct, whereas the injected construct was completely nonmethylated. We discuss methylation and other possible reasons for the observed differences in expression between injected and integrated copies of the β-actin/lacZ construct and for lacZ expression in only a subset of blastomeres in preimplantation embryos. © 1993 Wiley-Liss, Inc.     

The murine Cyp1a1 gene is expressed in a restricted spatial and temporal pattern during embryonic development

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.