Gene transfer and animal breeding

THERIOCENOLOGY

GENE TRANSFER AND ANIMAL BREEDING

R.0. Land and I. Wilmut AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Dryden Laboratory, Roslin, Midlothian, EH25 9PS, U.K.

ABSTRACT Genes can be isolated from an animal, multiplied and modified in the laboratory and transferred into animals of the same or of a different spec:.es. The effects of the additional gene depend upon the choice of control sequences and structural sequences in the transferred gene. In many cases transfer of a single gene (e.g. for one enzyme) would be expected to have little biological effect. However, t.hereare ot.her approaches. Increasing the concentration of a signal peptide, such as growth hormone, affects the activity of an entire metabolic pathway. This has been achieved by transfer of a 'construct' with control sequences that escape endogenous feedback systems for the hormone. Genes causing the production of novel proteins may modify animal performance (wool or milk) or produce proteins of use in human medicine (human blood clotting factors). It will be important to define both the beneficial and the deleterious effects of the gene and to compare the benefit gained with that available by conventional genetic selection. As gene expression depends upon the site of incorporation and number of copies of the gene, this assessment will be required for each transgenic line.

JANUARY

1987 VOL. 27 NO. 1

169

THERIOGENOLOGY

INTRODUCTION Animal breeding is the improvementof livestock for mankind by genetic change. It is presentlydependent upon the genetic variation which occurs betweenand within populationsand upon new variationwhich arises by natural, chance mutation. The animal breeder manipulates populations by selection to bring together favourable alleles in progressively ever more desirable combinations to give cumulative improvementin the chosen trait. Genetic research for animal breeding has concentrated on the recognition of genetic merit and on the determinationof appropriatestatisticalmethods to best manipulatethe genetic make-up by selectionboth among and within populations. This process, when applied rigorously,is very successfuland the rates of improvementwhich can be sustainedare summarisedin Table 1. The milk yield of cattle in the UK has increasedby more than 50% in the past 25 years, one half of this improvementis estimatedto be geneticand all of the evidenceindicatesthat yield could increasesimilarlyin the next 25 years. Table 1. Examplesof the rates of geneticchange (percentage of the mean per year) theoretically possibleby selectionand examplesof high rates achieved in selection experimentsand by industrya (taken from 1) trait

theoretical

experimental

industry

cattle sheep pigs

1.4 1.4 2.7

1.1 1.5

0.3 0.9 1.2b

poultry

3.2

4.1

6.5

carcassleanness cattle sheep pigs poultry

0.5 0.9 1.6 2.2

0.4

1.4

milk production

cattle

1.5

2.2

1.0

littersize

sheep pigs

2.1 3.0

1.2 1.0

2.9 1.5

egg production

poultry

2.1

1.1

1.7

growth rate

a After Smith 1984.

b

Focd conversionratio

This analysisis concernedin part with the genetic improvementof livestock by gene transfer. Given that several generations may be requiredto evaluatethe effectsof transferredgenes, and that economic merit may be increased by up to 2% per generation at present, a favourable effect of at least 10% is a reasonablethreshold for the

JANUARY

1987 VOL.

27 NO. I

THERIOGENOLOGY

magnitude of effect required for commercial gene transfer. transfer is also being used to make changes in animals possible by conventional genetic selection.

However gene that are not.

‘The genes which det.ennine t-he charact.eristics of individuals are transmit.ted from one generat.ion to t.he next. during t.he normal process of Both parents pass one half of their genes to each of their reproduction. offspring and the genes inherited are largely responsible for the determination of t.he characterist.ics of t.he new individual. Present knowledge of the structure of genes makes it. possible to identify individual genes as specific molecules of DNA, to remove them from one organism, to multiply (clone) them and modify them in the laboratory and to introduce them ‘to another organism. It is therefore possible in principle to cont.ribute artificially to the process of reproduct.ion and add specific genes t.o the normal process of inheritance bet.ween one generation and the next. The application of this knowledge to animal breeding depends upon the identification of useful genes, their isolation and, if necessary, modificat.ion and subsequent. incorporation to normal inheritance and t.he control of t.heir expression in the new, t.ransgenic animal.. Three met.hods of transfer have been pursued: 1) direct, physical injection to the male pronucleus, 2) retroviral aided introduction, and 3) a two step procedure based on t.he incorporat.ion of the chosen gene t.o the selection of cells t.hat, express a population of embryo st.em cells, of cells to the blast.ocyst the gene, and the subsequent. incorporation Most st.udies have been conducted with t-he laborat.ory mouse by (Fig. 1). direc:t injection when O-3% of injected embryos develop to transgenic adults in which t.he addit.ional gene is expressed. The rate of incorporation in domestic species has been lower than in mice. For sheep, 1 in 1,000 and 1 in 100 have been found to incorporate the gene injected (2; J.P. Simons, A.J. Clark and I. Wilmut, 1985, unpublished For pigs, Hammeret al (2) data I, but in neither case was it expressed. It. is to report that 20 of 2,000 injected embryos incorporated the gene. be expected t-hat success rates will improve as greater experience is aided gained in t-he manipulat.ion of farm animals. Retroviral incorporation has been achieved in mice (3), but. the viral sequences are Recent. observations suggest. that this failure occurs not expressed (4). because the viral control sequences do not initiate expression in the gene expression does occur in mammalian cells and that, by contrast, embryonic tissue if t-he transferred gene has an internal promoter (5). Germ line chimeras have been born following injection of embryo stem cells into the blastocyst cavity in mice (6). Gene transfer into embryo stem cells has been achieved by retroviral aided incorporation (5, 7). It remains to be confirmed t.hat. high levels of gene expression can be achieved in transgenic lines of mice established by t-his rout.e. One disadvantage of retroviral aided gene transfer into embryos and of t-he This is use of embryo stem cells is that. t.he animals are chimeras. Stem rarely the case following direct injection into the pronucleus. cell systems, possibly in combination with retroviral based met.hods to could be part.icularly important in increase the rate of incorporation, cattle where t,here would be considerable advant.ages of working with b1astocyst.s without t.he need for surgical collection or transfer of The technology of gene transfer is however addressed pronuclear embryos.

JANUARY

1987 VOL.

27 NO. 1

171

_

__.

Fiq.

I.

Three

rour.es

._

far the

production

animals

into

aided

of transaenic

incorporation blastocyst

retroviral

THERIOGENOLOGY

speciEically in t.he companion paper by J.P. Renard (8). The present subject is the role of this technology in animal breeding and the relevance of the new opportunities for the embryo transfer industry. The present t.echnology is the transfer oE single genes fused to a chosen promot.or. It is the addition of extra genes, not the replacement e:iist.ing of alleles. However, the possibility of treat.ing new “constructs” by combining novel control sequences t.o the st.ructural sequence of a desired gene is particularly import.ant. Such control sequences det.ermine t-he t.issues in which t.he gene will be expressed, t.he level of expression and the stages of development during which the gene will be active. Before assessing pot.ential strategies for t-he use of gene transfer it is helpful to consider the significance of single genes, including genes with major effects, in the context of the present understanding of gene action. THE IMPORTANCE OF SINGLEGENES The contribution of single genes may be assessed relative to, firstly, knowledge of single genes which are known to have useful effects in domest.ic livest.ock, secondly, knowledge of the effects of single genes in general in laboratory animals and thirdly, theoretical understanding of the effects of genes on the determination of the characteristics of animals. Domestic Livestock. There are now several examples of single genes which Gtase litt.er size in t.he sheep. The best documented js the Booroola but others are reported in Icelandic sheep (lo), and the gene (91, ‘Whether these are allelic or possibly Cambridge breed of sheep (11) . even t.he same allele is not. known. The only other examples of single genes affecting normally polygenic traits are the ‘halothane’ and double muscling genes of pigs and cattle respectively which increase the yield of lean meat. Not only are there few examples, it is also very relevant t.o not.e t.hat these genes have disadvantages as well as advant.ageous effects which limit. t.heir application. The double muscling gene increases calving difficulties; the ‘halothane gene’, while increasing t-he Lean yield, leads to an increased number of deaths as a result. of stress and a greater incidence of pale, soft exudat.ive meat. Even the Booroola gene is somet.imes considered to have too large an effect to be useful. The result. is that. none has widespread acceptance in commerce. On face value, the present evidence indicates that the molecular biologist is unlikely to find single genes wit.h favourable effects for t.ransgenesis

.

Laboratory Animals. The opportunity to st.udy single genes has been Maior aenes have areater in laboratorv animals than in farm animals. been detected by routine observations or during selection for a particular trait and their relevance to animal breeding has been Genes named ‘obese’, ‘adipose’ and discussed by Roberts & Smit.h (12). ‘diabetes’ all increase body weight to such an extent that their presence Unfort.unat.elv such qenes is u~ambiqouslv apparent in homozvqous animals. tend t-0 have-serious ill effect:s and may cause sterility or death. of single genes St.re.t.egies have been developed for the identification the wit-h rather smaller effect.s and also for selection t-0 limit

JANUARY

1987 VOL. 27 NO. 1

173

THERIOGENOLOGY

deleterious effects of major genes. The recent. identification of genes influencing embryonic development (13) and post-weaning growth (14) in mice lends hope to the conclusion that as we gain a greater understanding “t,he physiological and biochemical basis of quant i t.at ive traits, of individual loci with major effects may become more important” (12). Many of t.he major genes that have been st.udied exhibit a loss of function in comparison to the normal allele. In genetic analysis they are usually recessive. Transfer of such a gene would be expected to have no effect or little effect because of the presence of fully functioning alleles, Theory of Gene Action. The recessivity of most major genes and the scarcity of ones with favourable effects i.s consistent with biochemical knowledge of gene act.ion and of biochemical control t.heory as developed by Kacser and Burns (15) j n particular. They point out that t.he characteristics of animals are determined by the int.egrat.ion of the flux t.hrough many biochemical pathways and go on to show t.hat it. is unlikely that a change in the activity of any one gene will have a major effect on the rate of flux. They formalise the argument in terms of t.he sensitivity of the flux to a given change in the activity of a particular enzyme, the sensitivity coefficient. The sum of the coefficients for any one system is unity and for any one enzyme the coefficient. progressively decreases as the act.ivit.y of the enzyme j ncreases. These t.wo characteristics mean that addition of more of the product of existing genes is subject to the law of diminishing returns and that it increases the sensit.ivit.y coefficients of the other enzymes in the pathway. The alleviation of one biochemical deficiency immediately creates another. In conclusion, present. knowledge of practice, experiment and theory all indicate that. the introduct.ion of a single gene to affect. the rate of production of an enzyme in a complex, established pathway is unlikely to have a significant impact. on agricult.ure. Genetic selection as applied to farm animals has not identified many genes with major effects, but new strategies are being developed for the identification of useful single genes. of gene transfer is 9t. present, t-he framework for the application therefore likely to lie in the development of alternative approaches. Fortunately the same present knowledge also indicates where these are most likely to be found. STRATEGIES FOR MOLECULARMANIPULATION four broad approaches to the application of gene There are t.ransfer. either to manipulat.e a number of genes First I it is possible or to manipulate genes, such as those for protein hormones, that govern a by selection of novel control whole cascade of enzymes. Second, sequences it is possible +o escape endogenous feedback systems. Thirdly, there are situations in which products of single genes under normal cant rol are important. new products of value either to the Finally, animal or to man can be made by transgenic animals. In some cases more than one of these approaches has been used. Multiple Gene Clones and Hormones. One conclusion from control t.heory is that the elevation of the concentration of individual enzymes is unlikely

174

JANUARY

1987 VOL. 27 NO. 1

THERIOGENOLOGY

to i.ncrease t.he flux t.hrough a complex pathway. Equally however t.he theory indicates that if all enzymes in a pathway are simultaneously elevated the out.put would increase proportionally. This has been demonstrat.ed experimentally for tryptophan synthesis in yeast.. Plasmj ds carrying any one af the five principle enzymes of t.he pathway have been incorporated into yeast cells, but. were found not. t-o affect tryptophan synthesis significantly even with 10 to 50 plasmids (i.e. lo-50 times the concentration of the enzyme) per cell. Construction and incorporation of a plasmid with the genes for each of the fjve enzymes, however, showed a response in synt.hesis nearly proport.ional to t.he number of plasmids per cell (P. Niederberger, quoted by 16). Research with the pig shows the relevance of thjs knowledge to large animal breeding. Simultaneous selection for the level of four enzymf?s (G-6-P-DH, 6-P-G-DH, NADP-MDH and NADP-ICDH) in the NADPpathway led t.o marked changes in backfat in t.he same direct.ion as that of selection (17). After 8 generations of selection the high and low lines diffe--ed by 3.6 phenotypic standard deviation in backfat with a difference of 2.5 phenotypic standard deviation in enzyme activity (17). The effectiveness of hormones in causing significant changes in Wansgenj c animals is established. well Transgenic mice with supraphysiological levels of growt.h hormone (18) or growth hormone releasing factor (19) were markedly larger. Novel Control Sequences The first step is to ident.ify circumst.ances in which more hormone is required, the second is to identify a promotor which would ensure that the hormone was provided at. the appropriate stage of the life cycle, the third t-o prepare t.he fusion gene and the fourt.h t.o incorporate it t.o t.he of GH increases milk production, the attachment of genome. The injection the GH gene to the promotor for a gene normally switched on during lactat.ion might. then increase milk production. The original G ti-m etaUot_hionein t.ransgeni c mjce of Palmiter et. al (1982) showed t,he success of t.his approach (18). The GH gene was exprlessed in the liver and the plasma concentration of GH was increased way beyond that found with feedback cont.rol of the gene in t.he pituit.ary This creates t-he opport.unity to identify prom&or-st.ruct.ural gene gland. combinations which would ensure the controlled expression of the gene. si_nsl.e Genes Under Normal Control This approach has recei ved less attention, but mav have specific to or tolerance of some.parasitic infections is appl:.cat.ion. ‘-Resistance assol-iat.ed with certain alleles of t.he major histocompatibi1it.y complex. It may be possible to confer this advantage on lines of livestock by The nature of the product-s of cheese making transfer oE these alleles. is influenced in part by the protein in the milk. By t.ransfer of milk protein genes it may be possible to incorp0rat.e different. proteins in milk. Novel Product.s One clear introduction of

and particularly genes coding for

dramatic oppportunity is for the proteins important to human medicine

JA.NUARY 1987 VOL. 27 NO. 1

175

THERIOGENOLOGY

Diseases such as haemophilia are caused by the deficient action of (20). While recombinant bacterial insulin is now a medical single genes. reality, blood clotting factors cannot be produced in bacterial systems because of the inability of procaryotic cells to carry out post-translational processing of the protein. Both carboxylation and glycosilation are required for the synthesis of factors VIII and IX. It is now possible to envisage the fusion of human genes to, say, cattle milk protein genes so that the blood clotting factors would be produced by the manunarygland of cattle and could be extracted from milk. As a generalisation manunary production systems have several intrinsic advantages over bacterial systems - they are largely sterile: cattle and sheep maintain well buffered fermentation systems at much lower cost; the generation interval is much longer and stability of expression likely to be much greater. Such systems are therefore likely to be favoured in circumstances where markets are large enough to justify the initial investment in transgenesis in addition to those where there is a specific requirement for mammalian systems. Industrial enzymes would be one example. It has also been suggested that by transfer of the genes for the two bacterial enzymes involved, it may be possible to enable sheep to produce cysteine (21). As availibility of this essential amino acid determines the amount of wool produced, it may be that such transgenic sheep would produce more wool. FUTURE OPPORTUNITIES The sensitivity coefficient of an individual step in a biochemical pathway decreases as the concentration of enzyme is progressively increased. The inverse is that the sensitivity coefficent increases as the concentration of the enzyme is progressively decreased. Single genes which block the flux in a particular pathway are therefore likely to have major effects and this is compatible with the conclusion from the study of laboratory animals where most major genes are reductions in quantity or activity of an enzyme. The appropriate technology for further molecular manipulation would be to identify pathways in which feed-back acts to contain desirable phenotypic characteristics. Suppression of the feed-back would release the expression of the trait to better meet commercial requirements. A simple example of feedback constraint is the control of ovulation rate. Ovarian hormones control the release of gonadotrophins and hence the number of follicles recruited to ovulate. The identification of key feedback hormones and the reduction of their rate of synthesis would be expected to increase the number of ova shed. This is potentially an effective method to achieve genetic twinning in cattle. The development of this approach however depends on the identification of a key pathway and the reduction of the flux. The removal of genes is not presently envisaged, but approaches to reduce effective gene action have been identified. One is to change t.he existing genes by oligonucleotide mutagenesis (22). Errors in gene replication could be induced in vitro by the addition of oligonucleotides

176

JANUARY

1987 VOL. 27 NO. 1

THERIOGENOLOGY

with minor differences from the st.ruct.ural gene. The differences are incorporated and the gene is non-functional. This has not however been achieved in vivo and, further, it would be dependent upon t.he estabishment of cell line transgenics (Method 3, introduction), for it is in cell lines where the original mutation is most likely to be.induced. Anot.her approach is to neutralise the existing gene by int.roducing an antisense gene to code for the reciprocal of the mRNAfrom the gene to be reduced. The two RNAs t-hen hybridise to form a duplex so removing the original message from the mRNApool for t.ranslat.ion. The principle has been demonstrated by injecting antisense message to cells and reducing t.he rat.e of protein production (23). Is is a promising approach but has yet. t.o be effected. QUANTITATIVE ANDETHICALASPECTSOF UTILISATION Transgenesis essentially adds t.o the variation available. Once there, it may be subject. t.o normal selection or ident if ied and select.ed by a specific probe. The resources required to evaluate transgenic lives.tock will be very considerable. 3.A. Woolliams (1985, personal corranullication) pointed out that 200 dairy cat.tle would be required to assess whether any one of 5 constructs had a useful effect on milk product ion. Over four lactations this would represent 1,000 cattle Smith and his colleagues have studied the opportunities further, years. relative to the rate of change which can be achieved by conventional select.ion, and t-he populat.ion st.ruct.ures required to introduce transgenic stock to commercial production populations (24). They make the point that select.ion improves t.he overall economic merit of the stock whereas transgenesis changes but one biological component of only one performance charact.eristic, t.he t.ask can be seen to be daunting. 200 dairy catt.le would be needed to identify 1 of 5 useful construct.s - for milk product.ion. Many more would be required to test for disadvantageous effects on, t.raits such as fertility which have a higher coefficient of variation. Not only is the production of biomedical proteins a clear example of an obvious advant.age, t-he high value of t-he product. is such that. small unfavourable effects on traits such as food conversion efficiency or fert.ility would not be important. Welfare issues have always been important in animal production, and Genetic improvement has the this is particularly so at present. particular advantage that livestock products are better able to meet consumer requirements without the need for environmental physiological or Transgenesis will aid this process and be pharmacological int.ervent.ion. recognised by society as a contribution to the association between lnankind and domestic livestock. Embryo transfer companies must however be careful to ensure that the objectives of transgenic work are not misinterpreted. REFERENCES 1.

Knowledge for animal Land, R.0. Sot. Lond. B 310: 243-289 (1985).

JANUARY

1987 VOL.

27 NO.

1

breeding.

Phil

Trans.

R.

THERIOGENOLOGY

2.

Hannner,R.E., Pursel, V.G., Rexroad Jr., C.E., Wall, R.J., Bolt, D.J., Ebert,K.M., Palmiter,R.D. and Brinster,R.L. Productionof t.ransgenic rabbits,sheep and pigs by microinjection. Nature 315: 680-6831985).

3.

Jaenisch,R. Germ line integrationand Mendeliantransmissionof the exogenousMoloney leukemiavirus. Proc. Natl. Acad. Sci. USA 73, pp. 1260-1264(1976).

4.

Jaenisch,R., Jahner,D. Nobis,P., Simon, I., Lohler,J., Harbers, K. and Grotkupp, D. Chromosomal position and activation of retroviralgenomes insertedinto the germlinesof mice. Cell-24: 519-529 (1981).

5.

Stewart,C.L., Vanek, M. and Wagner, E.F. Expressionof foreign genes from retroviralvectors in mouse teratocarcinoma chimaeras. The EMBO Journal,4: 3701-3709(1985).

6.

Bradley,A., Evans, M., Kaufman,M.H. and Robertson,E. Formation of germ-line chimeras from embryo derived teratocarcinomacell lines. Nature,309: 225-256 (19841.

7.

Evans, M.J., Bradley, A., Kuehn, M.R. and Robertson,E.J. The ability of EK cells to form chimerasafter selectionof clones in G418 and some observationson the integrationof retroviralvector proviral DNA into EK cells. Cold SpringHarbor Sump. Quant. Biol. 50, pp. 685-689 (1985).

8.

Renard,

9.

Piper, L.R. and Bindon, B.M. The single gene inheritanceof the high litter size of the Booroola Merino. -In: Land, R.B. and Robinson, D.W., (ed) Genetics of Reproduction in Sheep. Butterworths, London,1985, pp. 115-125.

10.

Jonmundsson,J.V. and Adalsteinsson, S. Singlegenes for fecundity in Icelandic sheep. 7. In: Land, R.B. and Robinson, D.W., ted) Genetics of Reproductionin Sheep. Butterworths,London, 1985, pp. 159-168.

11.

Hanrahan, J.P. and Gwen, J.B. Variation and repeatability of ovulationrate in Cambridgeewes. Proc. Brit. Sot. Anim. Prod., Winter Meeting,March 1985, Paper No. 37 (1985).

12.

Roberts,R.C. and Smith,C. Genes with large effects- theoretical aspects in livestockbreeding. Proc. 2nd Wld. Congr. on Genetics Applied to LivestockProduction,Madrid, Oct. 1982, Vol. VI, pp. 420-438.

13.

Warner,C.M., Gollnick,S.O. and Golabard,S.B. Genetic controlof early embryonicdevelopmentby gene(s) in the MHC (H-Z complex). Proc. 10th Int. Cong. Anim. Reprod.& A.I. Urbana 4, XIII (1984).

178

Theriogenology.In press (19871.

JANUARY

1987 VOL. 27 NO. 1

THERIOGENOLOGY

14.

Bradford, G.E. and Famula, T.R. Evidence for a major gene for rapid postweaninggrowth in mice. Genet.Res. -44: 293-308 (1984).

15.

Kacser, H. and Burns, J.A. Moleculardemocracy:who shares the controls? Biochem.Sot. Trans.1: 1149-1160(1979).

16.

Kacser, H. Biochemicalpathways and their control. In: Bazin, M.J. and Prosser,J.I., (ed) MathematicalModels in Micsiology 3 In Press (1986). Physiological Models. CRC Press.

17.

Muller E. Physiologicaland BiochemicalIndicatorsof Growth and Composition.-In: Smith, C., King, J.W.B. and McKay, J.C., ted) ExploitingNew?&hnologies in Animal Breeding. oxford University Press. In Press (1986).

18.

Palimiter, R.D., Brinster,R.L., Hammer, R.E., Trumbauer,M.E., Rosenfield,M.G., Birnberg,N.C. and Evans,R.M. Dramaticgrowth of mice that develop from eggs microinjected with metallothionein-growth hormone fusiongenes. Nature 300: 611-615 (1982).

19.

Hannner,R.E., Brinster, R.L., Rosenfeld,M.G., Evans, R.M. and factor in Mayo, K.E. Expressionof human growthhormone-releasing transgenicmice results in increasingsomaticgrowth. Nature 315: 413-416 (1985).

20.

Lathe,R., Clark,A.J., Archibald,A.L., Bishop,J.O., Simons, P . and Wilmut, I. Novel products from livestock. In: Smith, C., King, J.W.B. and McKay, J.C., (ed) ExploitingNew Technologies in Animal Breeding: Genetic Developments. Oxford UniversityPress, Oxford. In Press (1986).

21.

Ward, K.A., Franklin, I.R., Murray, J.D., Nancarrow, C.D., Raphael, K.A., Rigby, N.W., Byrne, C.R., Wilson, B.W. and Hunt, C.L. Proc. 3rd Wld. Congr. on Genetics Applied to Livestock Production,Lincoln,Nebraska,1986,pp. 6-21.

22.

Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. and Insertion of DNA sequences into the Kucherlapati, R.S. human chromosomal -globin locus by homologous recombination. Nature317: 230-234 (1985).

23.

Izant,J.G. and Weintraub,H. Inhibitionof ThymidineKinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell -36: 1007-1015(1984).

24.

Smith,C., Meuwissen,T. and Gibson,J.P. On the use of trangenics in livestock improvement. Anim. Breeding Abstracts. In press (1986).

JANUARY

1987 VOL.

27 NO.

1

179