Efficiency Of Transgenesis Research Articles

Animal transgenesis: state of the art and applications

There is a constant expectation for fast improvement of livestock production and human health care products. The advent of DNA recombinant technology and the possibility of gene transfer between organisms of distinct species, or even distinct phylogenic kingdoms, has opened a wide range of possibilities. Nowadays we can produce human insulin in bacteria or human coagulation factors in cattle milk. The recent advances in gene transfer, animal cloning, and assisted reproductive techniques have partly fulfilled the expectation in the field of livestock transgenesis. This paper reviews the recent advances and applications of transgenesis in livestock and their derivative products. At first, the state of art and the techniques that enhance the efficiency of livestock transgenesis are presented. The consequent reduction in the cost and time necessary to reach a final product has enabled the multiplication of transgenic prototypes around the world. We also analyze here some emerging applications of livestock transgenesis in the field of pharmacology, meat and dairy industry, xenotransplantation, and human disease modeling. Finally, some bioethical and commercial concerns raised by the transgenesis applications are discussed.

Improving the generation of genomic-type transgenic mice by ICSI

Transgenes included in genomic-type constructs, such as yeast artificial chromosomes (YAC), P1-derived artificial chromosomes, or bacterial artificial chromosomes (BAC), are normally correctly expressed, according to the endogenous expression pattern of the homologous locus, because their large size usually ensures the inclusion of all regulatory elements required for proper gene expression. The use of these large genomic-type transgenes is therefore the method of choice to overcome most position effects, commonly associated with standard-type transgenes, and to guarantee the faithful transgene expression. However, in spite of the different methods available, including pronuclear microinjection and the use of embryonic stem cells as vehicles for genomic transgenes, the generation of transgenic animals with BACs and, particularly, with YACs can be demanding, because of the low efficiencies requiring extensive microinjection sessions and/or higher number of oocytes. Recently, we have explored the use of intracytoplasmic sperm injection (ICSI) into metaphase II oocytes as an alternative method for the generation of YAC transgenic mice. Our results suggest that the use of transgenic strategies based on ICSI significantly enhances the efficiency of YAC transgenesis by at least one order of magnitude.

Effect of Transgene Concentration, Flanking Matrix Attachment Regions, and RecA-Coating on the Efficiency of Mouse Transgenesis Mediated by Intracytoplasmic Sperm Injection1

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of approximately 510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.

Advances in transgenic rat production

Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.

Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.

402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng �L-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5%) was similar to that of non-injected control oocytes (67.8 ± 0.4%). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1%. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1% when the DNA concentration was increased to 40 ng µL−1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1%). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection

The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos.

378 EFFECT OF TRANSGENE CONCENTRATION AND FLANKING MATRIX ATTACHMENT REGIONS ON THE EFFICIENCY OF ICSI-MEDIATED TRANSGENIC MOUSE PRODUCTION

The efficiency of ICSI-mediated transgenesis can be influenced by many different factors. In this study, we have assessed the impact of transgene concentration and of the presence of transgene-flanking matrix attachment regions (MARs) on the efficiency of this procedure. Presented data include assays with three DNA constructs; a plasmid EGFP of 5.4 kb (pEGFP, Clontech Laboratories, Inc., Palo Alto, CA, USA); this plasmid EGFP construct flanked with two MAR elements (M-EGFP-M; 2.3 kb of the human �-interferon domain boundaries) (Guti�rrez-Ad�n et al. 2000 Transgenic Res. 9, 81-89); and a yeast artificial chromosome (YAC) of 520 kb. ICSI-mediated transgenesis was done in the B6D2 hybrid mouse strain as previously described (Moreira et al. 2004 Biol. Reprod. 71, 1943-1947), using two concentrations (1 and 15 ng/�L) of the M-EGFP-M transgene, three concentrations (1, 6, and 15 ng/�L) of the pEGFP transgene, and three concentrations (2, 3.6, and 5 ng/�L) of the YAC construct. Embryos were cultured in vitro until the 2-cell stage and transferred into Day 1 pseudopregnant females. Live offspring or Day 14 fetuses were analyzed by PCR for the detection of the genomic presence of the integrated transgene. The proportion of transgenics (PT) obtained with 2, 3.6, and 5 ng/�L of the YAC construct was 10, 21, and 13% respectively. A Z-test analysis of the data generated with this construct indicated that the number of transgenic offspring, when expressed as a proportion of injected ova (efficiency rate of the procedure, ER), increased with DNA concentration up to a threshold of 3.6 ng/�L, and suggested that higher concentrations of this transgene (and maybe of co-purifying contaminants) decreased the ER of the procedure. Interestingly, although much higher transgene concentrations were used for the pEGFP construct, this threshold was not reached. With an increase in transgene concentration (from 1 to 15 ng/�L), the PT increased (from 39 to 70%), as well as the ER of the procedure (from 3 to 7%). In addition, independently of the transgene concentration tested, when M-EGFP-M was used, the ER of the procedure and the PT observed were similar to the ones obtained with pEGFP. These results indicate that, regardless of the importance of other factors such as transgene size, purity, and copy number, the concentration to be used on ICSI-mediated transgenesis should be optimized for each construct, in order to maximize the PT among the liveborn offspring and the overall efficiency of the procedure. Our results also indicate that transgene-flanking MARs do not have a significant impact on the efficiency of this transgenesis method.

994. Gene Knockdown in Mice by Using Lentiviral Vectors Expressing Small Interfering RNA

Top of pageAbstract Many approaches have been described recently for generating loss-of-function phenotypes in eukaryotic systems using RNAi. Use of RNAi by means of long dsRNAs (>50bp) is limited in mammalian cells due to the induction of a nonspecific inhibitory response resulting from the interferon pathway. This limitation was overcome by the finding that small interfering RNAs (siRNAs) can specifically silence genes in mammalian cells without activating the nonspecific interferon pathway. We have designed lentiviral vectors expressing siRNAs to knock down the expression of specific genes. We have previously shown that a lentiviral vector capable of expressing siRNA specific for GFP (siGFP) was able to silence GFP expression in TgGFP mice upon transduction of fertilized mouse embryos. We have found that transgenesis of mouse embryos can be achieved both by zona pellucida removal prior to transduction and by microinjection of lentivirus particles under the intact zona layer. Efficiency of transgenesis was high using both methods, up to 80% and 100% respectively. However, survival of the treated embryos was low in the case of zona removal techniques (10-20%), compared to 40-60% for microinjection. We are currently using transgenesis by lentiviral vectors expressing small interfering RNAs to generate knockdown mice to study functional genomics.

Catalase in Manipulation Buffer Enhances the Developmental Competence of DNA-injected Embryos

To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P<0.01) in 100-units/ml catalase (81%) than those in 0 and 1,000 units/ml (56 and 65%). To investigate the ontogenetic ability of DNA-injected embryos, EGFP-injected embryos manipulated under 0 or 100 units/ml catalase were transferred separately to recipient mice. The proportion of fetuses derived from EGFP-injected embryos manipulated under 100 units/ml catalase (29%) was significantly higher (P<0.05) than that manipulated under 0 units/ml catalase (19%). Furthermore, the numbers of transgenic pups were 17 in 100 units/ml catalase and 14 in 0 units/ml catalase. The results of the present study indicate that scavenging reactive oxygen species during in vitro micromanipulation is beneficial for the development of DNA-injected embryos.

Compact intergenic regions of the pufferfish genome facilitate isolation of gene promoters: characterization of Fugu 3′-phosphoadenosine 5′-phosphosulfate synthase 2 ( fPapss2) gene promoter function in transgenic Xenopus

The highly compact nature of the pufferfish ( Fugu rubripes) genome renders it a useful tool not only for annotating coding regions within vertebrate genomes, but also for the identification of sequences important to gene regulation. Indeed, owing to this compaction it will be feasible in many instances to initiate analyses using entire intergenic regions when mapping gene promoters; a strategy that is very rarely feasible with the expanded genomes of other species. Stemming from our interest in studying promoters expressed in chondrocytes, we selected for study the intergenic region upstream of Fugu 3′-phosphoadenosine 5′-phosphosulfate synthase 2, fPapss2, a gene required for the normal development of cartilage extracellular matrix. Functional characterization of the entire fPapss2 5′ intergenic region was carried out by monitoring expression of the enhanced green fluorescent protein (EGFP) gene reporter in the developing cartilage of transgenic Xenopus laevis. By evaluating a series of 5′ intergenic region deletions we defined a minimal fPapss2 sequence of ∼300 bp that was essential for EGFP expression in tadpole cartilage. This functional analysis of an entire Fugu intergenic region, combined with the efficiency of Xenopus transgenesis, serves as a model for the rapid characterization of evolutionarily-conserved regulatory regions of other pufferfish genes.

Transposon-mediated enhancer trapping in medaka

We tested the Sleeping Beauty transposable element for its ability to efficiently insert transgenes into the genome of medaka ( Oryzias latipes), an important model system for vertebrate development. We show that the SB transposon efficiently mediates integration of a reporter gene into the fish germ line. In pilot experiments, we established 174 transgenic lines with a transgenesis efficiency of 32%. Transgenes are stably transmitted to, and expressed in, subsequent generations. Interestingly, the transgenic lines show novel expression patterns with temporal and spatial specificity at a rate of 12% (21/174), likely due to both, enhancing and silencing position effects. Furthermore, promoter-dependent GFP expression in injected fish embryos is tightly correlated with germ line transmission, facilitating easy selection of founder fish. Thus, the SB transposon/transposase system provides a highly efficient tool for transgenesis in general and for the generation of novel reporter gene expression patterns in particular.

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation.

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.

Metaphase II transgenesis

Since its advent in 1974, at least 11 methods have been developed to introduce potentially heritable exogenous DNA (transgenes; tgs) into mammals. These methods are now briefly reviewed in the context of a nascent method that has been demonstrated by microinjection of membrane-depleted sperm heads and tg DNA into metaphase II (mII) oocytes: mII transgenesis. The efficiency of mII transgenesis is at least as high as that of the well-established and prevailing alternative, pronuclear microinjection. Moreover, mII transgenesis promises to facilitate large tg delivery to assist with the generation of disease models and other paradigms in mammalian genome engineering.

Enhanced Green Fluorescence Protein (EGFP) as a Non-Invasive In Vivo Marker to Assess the Efficiency of Trangenesis and Nuclear Transplantation

Objective: We have successfully used mouse secondary polar bodies (2PBs) and female pronuclei (FPNs) to reconstruct viable embryos. However, factors attributing to the success of reconstruction remain unclear. The EGFP gene encoding a protein emitting green fluorescent light which can be easily observed by a fluorescent microscopy. In this study, we tried to established the method of integrating this exogenous DNA into the genome of the reconstructed embryos as a non-toxic biological marker to assess the efficiency of transgenesis and nuclear transplantation.

Efficiency of transgenesis using sperm-mediated gene transfer: generation of hDAF transgenic pigs

SINCE the beginning of this century, replacement of failing human organs with their animal counterparts has been an interesting topic of debate for writers and scientists. In the 1960s, prolonged survival after kidney transplantation from chimpanzee to human was obtained in the United States and Europe. Nevertheless, both the progressive improvement in surgical technique and in immunosuppressant therapy and the availability of cadaveric organs and living donation have reduced the interest in xenotransplantation. Because of the increasing requests for organs and the lack of donors to meet that need, xenotransplantation has become a reliable option again for temporary organ replacement (eg, of heart or liver) before definitive transplant. However, primates such as chimpanzees and baboons are expensive, can carry important zoonoses, and their use is burdened by ethical implications. A better choice for xenotransplantation might be offered by swine, which are closer to humans for anatomic and metabolic features. Discordant transplantation is associated with humoral hyperacute rejection, due to preformed antibodies and complement system activation (both classical and alternative pathway). This results in massive and irreversible vascular damage and cellular necrosis. Expression of the species-specific complement activation inhibitors could prevent this kind of rejection. Organs harvested from pigs transgenic for human decay accelerating factor (hDAF or CD55), membrane cofactor protein (MCP or CD46), and CD59 could be more efficiently grafted into human recipients. Therefore, a number of research teams, including our group, have generated pigs transgenic for human negative regulators of the complement cascade. This research has been aimed to generate hDAF transgenic swine with high efficiency and reproducibility. This goal has been reached through the innovative method of sperm-mediated gene transfer (SMGT), which was developed about 10 years ago by our group. Data presented in this paper show that hDAF-positive individuals can also be used as founders of stable lines of F1 generation transgenic pigs. MATERIALS AND METHODS hDAF Transgenic F1 Offspring Generation

A Low Frequency of Chromosomal Integration of Cloned DNA after Microinjection into the Cytoplasm of Fertilized Eggs of Chickens.

To evaluate the efficiency of transgenesis in chickens by the microinjection method, we have investigated the expression and integration of DNA which was microinjected into the cytoplasm of fertilized chicken eggs by histochemical staining, PCR and Southern analyses. We found that DNA injected into the cytoplasm was incorporated into the nucleus at an early cleavage stage and transcribed into mRNA. However, the DNA incorporated into the nucleus was rapidly lost during early development without integration into chromosomes. After 3 days of incubation, expression of the injected DNA was still observed in 38% of embryos, but the population of positive cells was very low in most of the embryos. PCR analysis indicated that a low amount of DNA was still retained in 35% of embryos after 7 days of incubation. However, in Southern analysis, injected DNA was detected in only one out of 76 embryos (1.3%). This embryo showed a single positive band after EcoRI digestion. The copy number was estimated to be about 0.2 copy per haploid genome from the intensity of the band, suggesting that chromosomal integration may have occurred after several cell divisions even if the detected DNA was integrated one. We obtained no case in which the injected DNA was integrated at the single cell stage, even though DNA was injected into the fertilized egg before pronucleus fusion. These results suggested that the DNA injected into the cytoplasm of the fertilized eggs may be incorporated into the cleavage nucleus at an early stage of development, but is rarely integrated into the chromosomal DNA. We concluded that the cytoplasmic injection method used in this study is a reliable method for expressing injected DNA transiently in early chicken embryos, but not an efficient method for generating transgenic chickens.

Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos.

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 degrees C, 15 min) and 4-6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

Production of proteins in the milk of transgenic livestock: problems, solutions, and successes

The milk of livestock can be modified dramatically by introducing foreign DNA into the germline. Exclusive expression of this DNA is ensured by the presence of regulatory sequences from mammary gland-specific genes. In sheep > 50% of the protein in milk can be encoded by a transgene and it appears that the foreign protein is additional to the normal complement of proteins. However, many technical hurdles (DNA configuration, low efficiency of transgenesis, and transgene stability) still prevent the routine use of this technology. In addition, such milk products have not yet received regulatory approval. These difficulties are not insurmountable. Transgenic methods can also be used to study the molecular basis of milk biogenesis. The effect on milk production in mice with all endogenous alpha-lactalbumin genes removed and in mice in which the murine genes were then replaced with human homologues is described. alpha-Lactalbumin, and consequently lactose, is essential for normal milk formation, and the human gene is expressed more efficiently than the murine gene.