Mice carrying either a deletion of the murine a-lactalbumin (a-lac) gene (null allele) or its replacement by the human a-lac gene (humanized allele) have been generated by gene targeting. Homozygous null females are a-lacdeficient, produce reduced amounts of thickened milk containing little or no lactose, and cannot sustain their offspring. This provides definitive evidence that a-lac is required for lactose synthesis and that lactose is important for milk production. Females homozygous for the humanized allele lactate normally, indicating that human a-lac can replace murine a-lac. Mouse and human a-lac expression was compared in mice heterozygous for the humanized allele. The human gene expressed "15-fold greater mRNA and "14-fold greater protein than the mouse, indicating that the major determinants of human a-lac expression are close to, or within, the human gene and that the mouse locus does not exert a negative influence on a-lac expression. Variations in a-lac expression levels in nondeficient mice did not affect milk lactose concentration, but the volume of milk increased slightly in mice homozygous for the humanized allele. These variations demonstrated that a-lac expression in mice is gene dosage dependent.Milk is a complex mixture of proteins, lipids, carbohydrates, and inorganic components, the composition of which varies widely between species. The whey protein a-lactalbumin (alac) is thought to influence milk carbohydrate and fluid content through its role as a component of the lactose synthase complex (1). Lactose provides a major osmotic component of milk and determines milk volume by influencing water influx. It has been proposed that the lactose content of milk is directly related to the quantity of a-lac present (2), but a causative relationship has yet to be demonstrated in vivo.We have used gene targeting in embryonic stem cells to produce null and replacement alleles at the murine a-lac locus (3). Mice in which the murine a-lac gene has been completely deleted (null allele) provide a definitive test of the role and importance of a-lac in lactation and mammary physiology. Mice in which the murine a-lac gene has been replaced by the human gene have been used (i) to test whether human a-lac can functionally substitute for mouse a-lac, and (ii) to investigate determinants of a-lac gene expression, since humans and mice produce markedly different amounts of milk a-lac. MATERIALS AND METHODSMouse Lines. Derivation of mice bearing null and humanized a-lac alleles has been described (3).RNA Analysis. Total RNA was prepared from abdominal mammary glands of female mice 5-6 days postpartum. RNase protection analysis used a [32P]CTP-labeled antisense RNA probe transcribed from a 455-bp HindIII-Bal I mouse a-lac fragment (see Fig. 4A) cloned in pBluescript KS. Experimental conditions were as recommended by Promega.Milk Composition and Yield Analysis. Milk samples were collected between days 3 and 7 of lactation. Milk fat content was measured as described by Fleet and Linzell (4). Defatted milk wa...

The mouse a-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse a-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human a-lactalbumin. The MATERIALS AND METHODSDNA cloning and constructs. The mouse a-lactalbumin gene was isolated from a library of strain 129 mouse genomic DNA (Stratagene) by hybridization with a mouse a-lactalbumin probe generated by PCR. Similarly, the human gene was isolated from a human genomic library (Stratagene) by hybridization with a human a-lactalbumin PCR fragment. Two overlapping X clones were isolated and characterized for each gene. A restriction map of the cloned mouse a-lactalbumin locus is shown in Fig. 1. The identity of cloned genomic fragments was verified by comparison of partial DNA sequences (data not shown) with the published human genomic (9) and mouse cDNA (35) a-lactalbumin sequences.MALHT. The MALHT (mouse alpha-lactalbumin, HPRT, TK) targeting vector shown in Fig. 1

Producing transgenic cattle by microinjection of DNA into pronuclei has been inefficient and costly, in large part because of the cost of maintaining numerous nontransgenic pregnancies to term. We designed a system for early identification of transgenic embryos in which biopsies of embryos were assayed by polymerase chain reaction for presence of the transgene before embryo transfer. A total of 2555 embryos were microinjected with one of two DNA constructs. Of the 533 embryos biopsied, 112 were judged to be potentially transgenic and were transferred nonsurgically to recipients, resulting in production of 29 putative transgenic fetuses. One fetus and one calf (7% of offspring) were subsequently shown to be definitively transgenic. The calf was transgenic for a chicken c-ski cDNA, and several months after birth developed dramatic muscular hypertrophy followed by muscle degeneration. This phenotype was associated with expression of high levels of mRNA from the transgene.

Expression vectors derived from pSV2gpt (1) are commonly used to direct abundant expression of genes from the simian virus 40 (SV40) early promoter. However, use of this type of vector is complicated by the paucity of restriction enzyme sites available for insertion of fragments. SVpoly is a simple vector designed to provide convenient restriction sites to facilitate the generation and manipulation of constructs for expression in mammalian cell culture, or transgenic animals. SVpoly consists of a polylinker flanked by the SV40 early promoter and the SV40 late polyadenylation signal in a small plasmid which grows to high copy number in a bacterial host. Seven unique restriction sites are available in the polylinker for insertion of the sequence to be expressed. Unique restriction enzyme sites 5' of the promoter and 3' of the polyadenylation site make it simple to exchange either of these for others if desired. We have used SVpoly and derivatives carrying different promoters to express selectable marker genes, antisense RNA and protein coding sequences in a wide variety of cell types. We find that the amount of expression by SVpoly is comparable to that by pSV2. SVpoly was constructed as follows: The SV40 promoter was derived from pU (2) as a BamHI HindIII fragment and corresponds to a PvuH HindIlI fragment (positions 270 and 5171) of SV40. The polylinker is a HindIl BamHI fragment (positions 78 and 29) of pPolyIII (3), see sequence below. The SV40 late polyadenylation signal was derived from pSVL (4) as a BamHI Sall fragment and corresponds to a BamHI BclI fragment (positions 2533 and 2770) of SV40 coupled to a BamHI SalI fragment (positions 375 and 651) of pBR322. The beta lactamase gene and bacterial origin of replication are provided by pPolyIII (3) from XhoI to BglIH (positions 19 and 131). Bgl ACKNOWLEDGEMENT We acknowledge the financial support of the Colorado State University Experimental Station Fund (Grant # 1-56261).

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The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human alpha-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus.